EP3759248A1 - Procédé pour l'amplification d'un acide nucléique avec spécificité améliorée - Google Patents

Procédé pour l'amplification d'un acide nucléique avec spécificité améliorée

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Publication number
EP3759248A1
EP3759248A1 EP19712690.7A EP19712690A EP3759248A1 EP 3759248 A1 EP3759248 A1 EP 3759248A1 EP 19712690 A EP19712690 A EP 19712690A EP 3759248 A1 EP3759248 A1 EP 3759248A1
Authority
EP
European Patent Office
Prior art keywords
sequence
primer
amplification
oligonucleotide
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19712690.7A
Other languages
German (de)
English (en)
Inventor
Dmitry Cherkasov
Christian GRUNWALD
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AGCT GmbH
Original Assignee
AGCT GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE102018001586.7A external-priority patent/DE102018001586A1/de
Priority claimed from EP18195312.6A external-priority patent/EP3530754A1/fr
Application filed by AGCT GmbH filed Critical AGCT GmbH
Publication of EP3759248A1 publication Critical patent/EP3759248A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present invention relates to a method for the amplification of nucleic acids with improved specificity.
  • PCR nucleic acid chains plays a central role in biotechnology today.
  • Methods such as PCR have significantly advanced both the research landscape and industrial application fields such as diagnostics and the food industry.
  • technologies such as sequencing, real-time detection, microarray technology, microfluidic management etc.
  • Other amplification techniques such as isothermal amplification techniques have also been developed. Their use was especially intended for POCT (point-of-care testing).
  • POCT point-of-care testing
  • the PCR does not control the amplified sequence segments located between the primers.
  • the primer binding is in the focus of optimizations of PCR methods.
  • the initiation of the synthesis of major products and by-products e.g. by non-specific binding or extension of the primer.
  • the unspecifically extended primer is read off as a template, which generally leads to the formation of a complete primer binding site.
  • a transmission of erroneous sequence information from one synthesis cycle to the next occurs, which in sum leads not only to the initial formation, but above all to the exponential multiplication of by-products.
  • Such side reactions can lead to the exponential growth of fragments which interfere with the main reaction (amplification of a target sequence) or lead to interferences in subsequent steps of the analysis.
  • Such by-products typically include left and right primer sequences so that their amplification can occur in parallel with the main reaction.
  • such by-products include another nucleic acid target sequence.
  • the specificity of the PCR amplification is achieved by optimizing the primer binding to target sequences. In this case, for example, additional oligonucleotides can be used, which partially bind to the primer and thus can competitively participate in primer binding to other nucleic acid chains.
  • Such probes typically bind to a sequence portion of the primer and release a single-stranded portion that allows the primer to bind to the target nucleic acid and initiate a synthesis reaction.
  • Primer matrices Mismatches can be competitively displaced by such oligonucleotides, which improves the specificity of the initiation.
  • Such additional oligonucleotides do not interact with the nucleic acid chain to be amplified in portions between the two primers. However, due to a molar excess of the primers, non-specific interactions of primers with templates may occur during amplification, resulting in the formation of a fully functional primer binding site as a result of the synthesis of a complementary strand of the by-product.
  • the side reactions in a PCR increase with the number of synthetic cycles performed, often up to 40 cycles of PCR being performed.
  • other methods can be connected, e.g. Detection, l / bra / y production, cloning, sequencing, etc. These methods can only tolerate to a certain extent the by-products or sequence deviations produced during PCR amplification. In general, the specificity of such methods is also influenced by the problem of side reactions during a PCR.
  • Another object of the invention is to provide a combination of an amplification method with a PCR amplification, wherein the signal acquisition (monitoring or detection), or the amplification of target sequences first by means of controlled amplification using a controller oligonucleotide and corresponding specific primer Sets is carried out and then continued by means of PCR, in particular using PCR-specific primer.
  • Another object of the invention is to reduce the number of PCR synthesis cycles necessary to obtain a sufficient amount of PCR products.
  • Another object of the invention is to provide a novel enzymatic method and components for the synthesis of nucleic acid chains, as well as the amplification of nucleic acid chains, in which a continuous (on-line) signal detection is provided, wherein the signal acquisition (Monitoring / Detection) by sequence-specific oligonucleotide probes.
  • a first aspect of the invention relates to a method for amplifying a nucleic acid, comprising the steps:
  • Hybridizing a first primer oligonucleotide (P1.1) to a nucleic acid to be amplified having a target sequence wherein the first primer oligonucleotide (P1.1) comprises the following ranges:
  • a first region (P1 .1 .1) which can bind sequence-specifically to a region of the nucleic acid to be amplified, wherein the region of the nucleic acid to be amplified comprises at least the 5 ' terminus of the target sequence or in the 5 ' direction of the Target sequence is located;
  • a second region (P1 .1 .2) connected to the 5 'end of the first region or connected via a linker, which second region can be bound by a controller oligonucleotide and for the polymerase used chosen reaction conditions remains essentially unkopiert;
  • a first primer extension product (P1 .1 -Ext) which comprises, in addition to the first primer oligonucleotide (P1 .1), a synthesized region which is substantially is complementary to the nucleic acid to be amplified or to the target sequence, wherein the first primer extension product and the nucleic acid to be amplified are present as a double strand;
  • Binding of a controller oligonucleotide (C1 .1) to the first primer extension product (P1 1 -Ext), wherein the controller oligonucleotide (C1 .1) comprises the following ranges:
  • a second region which is substantially complementary to the first region of the first primer oligonucleotide (P1 .1), and A third region (C1 .1 .3) that is substantially complementary to at least a portion of the synthesized region of the first primer extension product (P1 .1 -Ext); and
  • controller oligonucleotide (C1 .1) does not serve as a template for primer extension of the first primer oligonucleotide (P1 .1) and the first controller oligonucleotide (C1 .1) to the first and second regions of the first primer extension product (P1 .1-Ext) under displacement (of the region complementary to the first and second regions) of the nucleic acid to be amplified;
  • Hybridizing a second primer oligonucleotide (P2.1) to the first primer extension product wherein the second primer oligonucleotide (P2.1) comprises a region (P2.1.1) which is sequence-specifically attached to the synthesized region of the first primer Extension product (P1 .1 -ext) which is at least complementary to the 5 ' terminus of the target sequence or located in the 3 ' direction thereof; Extension of the second primer oligonucleotide (P.2.1) by the first polymerase to obtain a second primer extension product (P2.1 -Ext), which in addition to the second primer oligonucleotide (P2.1) comprises a synthesized region which is substantially identical to the nucleic acid or the target sequence to be amplified, wherein the first primer extension product (P1 .1-Ext) and the second primer extension product (P2.1) form a first double-stranded amplification product; and
  • Extension of the third primer oligonucleotide (P3.1, P3.2) by a second polymerase to give a third primer extension product (P3.1 / 2-Ext) which is next to the third primer oligonucleotide (P3.1, P3. 2) comprises a synthesized region that is substantially complementary to the second primer extension product (P2.1-Ext) or to the target sequence, wherein the second primer extension product (P2.1) and the third primer extension product (P3.1) present as a double strand;
  • a fourth primer extension product (P4.1 / 2-Ext) which comprises, in addition to the fourth primer oligonucleotide, a synthesized region expressed in the Substantially complementary to the first primer extension product (P1 1 -Ext) or identical to the target sequence, wherein the first primer extension product (P1 1-Ext) and the fourth primer extension product (P4.1 / 2-Ext) are in duplex;
  • one aspect of the invention may also be formulated as a method of amplifying a nucleic acid ( Figures 1, 55-56), wherein a sample comprising a first nucleic acid polymer comprising a first target sequence M1 [and the M1 (reverse) complementary sequence M1 ' ], where M in the 5'-3' orientation comprises in direct sequence the sequence segments M1 .5, M1 .4, M1 .3, M1 .2 and M1 .1,
  • a first template-dependent nucleic acid polymerase in particular a DNA polymerase, and also substrates of the template-dependent nucleic acid polymerase (in particular ribonucleoside triphosphates or deoxyribonucleoside triphosphates) and suitable cofactors, such as Mg salts,
  • a first (right) oligonucleotide primer P1 .1 which in the 5'-3 'orientation directly comprises the sequence segments P1 .1 .2 and P1 .1 .1, P1 .1 .1 having a complement to M1 .1 [ hybridizing] sequence [can bind in a substantially sequence-specific manner] and the P1 .1 .2 sequence portion can not bind to M1 [or a sequence immediately adjacent to M1 .1 with respect to the M1 sequence in 3 '], and P1 .1 (in particular in section P1 .1 .2) modified nucleotide building blocks, so that P1 .1 .2 can not serve as a template for the activity of the first template-dependent nucleic acid polymerase; and iii.
  • a second (left) oligonucleotide primer P2.1 which is (substantially) identical to M1 .5 [and sequence-specifically the reverse complementary sequence of M1 .5 on the opposite strand of M1 or the extension product of P1 .1, P1 1 -ext, there as P1 .1 denotes E1, can bind];
  • C1 .2 in C1 .2.1 comprises modified nucleotide building blocks such that C1 .2.1 can not serve as template for the activity of the template-dependent nucleic acid polymerase;
  • Sequence section 3.1 .1 comprises [or consists essentially of 3.1 .1] that is (reverse) complementary to M1 .1 of M1 [can bind complementarily to P2.1 -hex],
  • Sequence Section 4.1 .1 comprises [or consists essentially of 4.1 .1] that is identical or substantially identical to M1 .5 of M1 [can complementally bind to P1 1 -ext], vii. a second template-dependent nucleic acid polymerase, in particular a DNA
  • Polymerase and optionally substrates of the template-dependent nucleic acid polymerase (especially ribonucleoside triphosphates or deoxyribonucleoside triphosphates) and suitable cofactors.
  • substrates of the template-dependent nucleic acid polymerase especially ribonucleoside triphosphates or deoxyribonucleoside triphosphates
  • suitable cofactors especially ribonucleoside triphosphates or deoxyribonucleoside triphosphates
  • a first primer extension product P1 .1-Ext. obtained in 5'-3 'orientation in addition to the sequence regions P1 .1 .2 and P1 .1 .1 comprises a synthesized region in the 5'-3' orientation, the sequence sections P1 .1 E4, P1 .1 E3 , P1 .1 E2 and P1 .1 E1, where P1 .1 E4 to the sequence section M1 .2 of the target sequence M1, P1 .1 E3 to M1 .3, P1 .1 E2 to M1 .4 and P1 .1 E1 to M1 .5 is substantially complementary.
  • a second primer extension product P2.1 -Ext is obtained, which in addition to the sequence area P2.1 .1 a synthesized area P2.1 -Ext. which is substantially identical to the target sequence M1 in the area adjacent to M1 .5 in FIG. 3 '.
  • the reaction conditions and / or the lengths or melting temperatures of P1 .1 .2 and possibly M1 .4, M1 .3, M1 .2 and M1 .1 of the first amplification step are selected such that P1 1-Ext with M1 or with P2.1 -xt can form a double strand, and P1 .1 -Ext can form a double strand with C1 .2 and the formation of the double strand from P1 1 -Ext with C1 .2 compared to the formation of the double strand from P1 1-Ext with P2 .1 text is preferred.
  • the invention may also be formulated as a method of amplifying a nucleic acid wherein a sample comprising a first nucleic acid polymer comprising a first target sequence M1 is contacted in a first amplification step with the following components:
  • a first template-dependent nucleic acid polymerase in particular a first DNA polymerase, and substrates of the template-dependent nucleic acid polymerase (in particular ribonucleoside triphosphates or deoxyribonucleoside triphosphates) and suitable cofactors,
  • a first (right) oligonucleotide primer P1 .1 comprising, in the 5'-3 'orientation, in direct sequence the sequence segments P1 .1 .2 and P1 .1 .1, at least 3 ' segment of P1 .1 .1 can bind a section of M1 (essentially) sequence-specifically and the sequence section P1 .1 .2 can not bind to M1, and
  • P1 .1 (in particular in section P1 .1 .2) comprises modified nucleotide units, so that P1 .1 .2 can not serve as template for the activity of the first template-dependent nucleic acid polymerase;
  • a second (left) oligonucleotide primer P2.1 comprising at least one 3 ' segment which is (substantially) identical to one in 5' of the binding site of P1 .1.
  • M1 is essentially identical, or a binding site can bind the reverse complementary sequence on the opposite strand of M1 or the extension product of P1 .1 [also referred to as P1 .1-Ext, as P1 .1 E1];
  • a controller oligonucleotide C1 .2 which in the 5'-3 'orientation comprises the sequence sections C1 .2.3, C1 .2.2 and C1 .2.1 in direct sequence:
  • a third region (C1 .2.3) of the controller oligonucleotide which is substantially complementary to at least part of the synthesized region of the extension product of the first primer (P1 1 -Ext) formed in an amplification reaction, in other words the third region being identical to one Is the portion of M1 which is located at 5 'of the binding site of the first primer with respect to the polarity of the strand read [and is read first upon initiation of the polymerase of P1 .1, ie the third region of the controller is at least part of the polymerisation product of the first first primer [P1 .1 -xt],
  • a second region (C1 .2.2) of the controller oligonucleotide which is substantially complementary to the first region of the first primer oligonucleotide (P1 .1) (and identical at least to a portion of the binding site of the first primer on the target sequence M 1) and
  • a first region (C1 .2.1) of the controller oligonucleotide attached to the sequence section P1 .1 .2 of the first primer or the 5 'terminal region of the first primer can bind to an extension product of the first primer (P1 .1-Tex) formed in an amplification reaction, and
  • C1 .2 comprises nucleotide building blocks modified at least in C1 .2.1 C1 .2.3 so that C1 .2.1 can not serve as template for the activity of the template-dependent nucleic acid polymerase;
  • the sample is incubated with the above components under conditions permitting a first amplification to form amplification products comprising primer extension products of the first oligonucleotide primer (P1.1-Tex) and the second oligonucleotide primer (P2.1-Ext) containing the target sequence M or at least their proportions and corresponding complementary sequences include; and wherein the sample is subsequently or simultaneously brought into contact with the following components in a second amplification step:
  • Sequence section 3.1 .1 comprises [or substantially consists of 3.1 .1] which can bind (reversely) complementarily to the amplification product formed in the first amplification step, in other words: the sequence-specifically the 3 'terminal region of the extension product of the extension product of the first amplification reaction can bind second primer,
  • Sequence section 4.1 .1 comprises [or substantially consists of 4.1 .1] which can bind (reversely) complementarily to the amplification product formed in the first amplification step, in other words: the sequence-specifically the 3 'terminal region of the extension product of the extension product of the first amplification reaction can bind first primer,
  • G a second template-dependent nucleic acid polymerase, in particular a DNA
  • Polymerase and optionally substrates of the template-dependent nucleic acid polymerase (especially ribonucleoside triphosphates or deoxyribonucleoside triphosphate) and suitable cofactors.
  • substrates of the template-dependent nucleic acid polymerase especially ribonucleoside triphosphates or deoxyribonucleoside triphosphate
  • suitable cofactors especially ribonucleoside triphosphates or deoxyribonucleoside triphosphate
  • the sample is incubated with the above components under conditions permitting a second amplification to form amplification products comprising primer extension products of the third oligonucleotide primer (P3.1-Ext) and the fourth oligonucleotide primer (P4.1-Ext) containing the target sequence M or at least their proportions and corresponding complementary sequences.
  • P3.1-Ext primer extension products of the third oligonucleotide primer
  • P4.1-Ext fourth oligonucleotide primer
  • kits comprising the following components:
  • P1 .1 .2 comprises modified nucleotide units, so that P1 .1 .2 can not serve as template for the activity of a template-dependent nucleic acid polymerase;
  • oligonucleotide primer P2.1 which can sequence-specifically bind a sequence on the opposite strand of the sequence bound by the first oligonucleotide primer
  • C1 .2.3 can bind to the polymerization product of the primer P1 1-Ext]
  • ii. C1 .2.2 is complementary to P1 .1 .1 (and identical to M1 .1)
  • iii. C1 .2.1 is complementary to P1 .1 .2
  • C1 .2 in C1 .2.1 comprises modified nucleotide building blocks such that C1 .2.1 can not serve as template for the activity of the template-dependent nucleic acid polymerase;
  • a first template-dependent nucleic acid polymerase in particular a DNA polymerase, and optionally substrates of the template-dependent nucleic acid polymerase (in particular ribonucleoside triphosphates or deoxyribonucleoside triphosphates) and suitable cofactors such as Mg salts;
  • a third (right) oligonucleotide primer P3.1 which is different from the first oligonucleotide primer P1 .1, in particular does not have its section P1 .1 .2, and which at the 3 'terminus comprises a sequence section 3.1 .1 [or substantially from 3.1. 1] which is (reverse) complementary to [Section M1.1] of the target sequence [M1];
  • a fourth (left) oligonucleotide primer P4.1 which comprises at the 3 'terminus a sequence segment 4.1 .1 [or consists essentially of 4.1 .1] which can sequence-specifically bind a sequence on the opposite strand of the sequence linked by the third oligonucleotide primer;
  • G a second template-dependent nucleic acid polymerase, in particular a DNA polymerase, and optionally substrates of the template-dependent nucleic acid polymerase (in particular ribonucleoside triphosphates or deoxyribonucleoside triphosphates) and suitable cofactors.
  • substrates of the template-dependent nucleic acid polymerase in particular ribonucleoside triphosphates or deoxyribonucleoside triphosphates
  • a further aspect of the invention relates to a method for the amplification of a nucleic acid, wherein a sample which contains a nucleic acid comprising a first target sequence M1, in sequence 5'-3 'in immediate sequence, the sequence sections M1 .5, M1 .4, M1 .3, M1 .2 and M1 .1 is brought into contact in a first amplification step with the following components:
  • a first template-dependent nucleic acid polymerase in particular a DNA polymerase, and substrates of the template-dependent nucleic acid polymerase, a first (right) oligonucleotide primer P1 .1, in sequence 5'-3 'in immediate sequence, the sequence sections P1 .1 .2 and P1 .1 .1, wherein P1 .1 .1 has a [hybridizing] sequence complementary to M1 .1 and the sequence segment P1 .1 .2 can not bind to M1, and
  • P1 .1 (in particular in section P1 .1 .2) comprises modified nucleotide units, so that P1 .1 .2 can not serve as template for the activity of the first template-dependent nucleic acid polymerase;
  • oligonucleotide primer P2.1 which is (substantially) identical to M1 .5;
  • a controller oligonucleotide C1 .2, which in the 5'-3 'orientation comprises the sequence sections C1 .2.3, C1 .2.2 and C1 .2.1 in direct succession, C1 .2.3 being identical to a section M1 .2 of M1, which is in the 5 'direction of M1 .1, C1 .2.2 is complementary to P1 .1 .1 (and identical to M1 .1) and C1 .2.1 is complementary to P1 .1 .2,
  • C1 .2 in C1 .2.1 comprises modified nucleotide units such that C1 .2.1 can not serve as template for the activity of the template-dependent nucleic acid polymerase,
  • a. comprises a sequence section which
  • ii. is complementary to a sequence of M1 located on the sequence sections M1 .3 and M1 .4,
  • an aspect of the invention may be formulated as a method comprising the following steps:
  • a first amplification system comprising a first primer oligonucleotide, a second primer oligonucleotide, a controller oligonucleotide, a first DNA polymerase, and first polymerase (dNTPs) substrates and a suitable buffer solution
  • amplification system comprising a third primer oligonucleotide, a fourth primer oligonucleotide, a second thermostable polymerase, and second polymerase (dNTPs) substrates and suitable buffer solution.
  • a detection system is furthermore provided, insofar as the subject matter of the invention also encompasses the detection by the first and / or the second amplification system of amplified nucleic acid fragments, in particular with probes.
  • the reaction conditions of the first amplification used comprise at least one temperature step which permits hybridization and template-dependent primer extension of both primers of the first amplification system and at least one temperature step in which the first primer extension product of the second primer extension product participates of the controller oligonucleotide is separated.
  • the reaction conditions used do not allow spontaneous separation of the first primer Extension product from the second primer extension product in the absence of the controller oligonucleotide.
  • the first amplification is carried out until the desired amount of the first amplification product 1 .1 has been synthesized.
  • the properties of the components of the respective amplification system, the nucleic acid fragment comprising a first target sequence, and the reaction conditions used determine the course of the two amplification reactions.
  • the first amplification takes place with the assistance of the controller oligonucleotide.
  • the separation of the two synthesized primer extension products takes place at least in part depending on the sequence of the first primer extension product and its complementarity with the sequence of the controller oligonucleotide.
  • a second amplification fragment is amplified substantially without the involvement of the controller oligonucleotide.
  • both desired amplification products (amplification fragment 1 .1 and amplification fragment 2.1) can be formed, as well as their intermediates (intermediates).
  • the composition of the intermediates depends essentially on the provided components of the first and second amplification systems.
  • Fig. 1 shows schematically the components of certain embodiments of the amplification method according to the invention in a heterogeneous format; A) of the first amplification system; B) of the second amplification system.
  • Fig. 2 shows schematically the components of certain embodiments of the first
  • FIG. 3 shows a temperature profile of certain embodiments of the method according to the invention in heterogeneous format; An aliquot is transferred after amplification 1 .1 in amplification 2.1. This results in a dilution of the first amplification fragments.
  • FIG. 4 shows a temperature profile of certain embodiments of the method according to the invention in heterogeneous format; Components of the second amplification system are added. There is no significant dilution of fragments generated by A-1.1.
  • Fig. 5 shows the components of certain embodiments of the amplification method according to the invention in a homogeneous format; A) of the first amplification system; B) of the second amplification system.
  • Fig. 6 to 1 1 shows temperature profiles of embodiments of the invention
  • Fig. 12 schematically shows (A) the structure of a first primer oligonucleotide; (B) the complementary binding of the first primer to the nucleic acid chain to be amplified; (C) the complementary binding of the first primer to the nucleic acid chain to be amplified and the extension of the primer.
  • Fig. 14 shows schematically the synthesis of the second primer extension product
  • Fig. 15 shows schematically the synthesis of the third and fourth primer extension products
  • P3.1 -ext part 1 extension product of P3.1 synthesized on P2.1-Ext as template (intermediate);
  • P4.1 -Ext part 1 extension product of P4.1 synthesized on P1.1 -Ext as template (intermediate);
  • P3.1 -Ext extension product of P3.1 synthesized using P4.1 Ext part 1 or at P4.1 -Ext as template;
  • P4.1-Ext extension product of P4.1 synthesized using P3.1 -xt part 1 or as template at the P3.1 -xt
  • Figs. 27 to 31 schematically show the components of various embodiments of the first amplification system; (A) components of the first amplification system; (B) Components of the second amplification system.
  • Figures 27-29 the fourth primer is identical to the second primer, the third primer is different from the first primer. Its binding may be shifted relative to the first primer.
  • Fig. 30 the fourth primer is different from the second primer. 3 ' end of the fourth primer is optionally shifted with respect to the 3 ' end of the second primer.
  • the fourth primer comprises copyable sequence segments which are not complementary to the target sequence or do not bind to P1 .1-Ext.
  • FIG. 31 The third primer comprises copyable sequence segments which are not complementary to the target sequence or do not bind to P2.1-Ext.
  • Fig. 32 shows schematically (A) a first primer oligonucleotide and (B) a nucleic acid to be amplified (P1 1 -Ext and P2.1 -Ext);
  • Fig. 33 shows schematically the strand displacement by a controller oligonucleotide
  • Fig. 34 shows schematically the interactions between components in a reaction of the method according to the invention; The intermediate step of separating the second primer extension product from the double-stranded complex consisting of the first primer extension product and controller oligonucleotide is apparent.
  • Fig. 35 shows schematically an amplification by simultaneous synthesis of two strands and separation of the synthesized amplification fragments
  • Figs. 36 to 49 show the results of example experiments.
  • Figures 50 and 51 schematically show certain embodiments of the preparation of a starting nucleic acid chain starting from genomic DNA.
  • FIGS. 52 to 54 schematically show certain embodiments of the ampification according to the invention.
  • Figures 55 to 57 schematically show certain embodiments of the topography of the components of the first amplification system and the second amplification system.
  • FIGS. 58 to 59 schematically show certain embodiments of the possible localization regions for oligonucleotide probes:
  • Fig. 60 shows schematically certain embodiments of the localization of probe segments, which can bind predominantly complementary to the third (and possibly the first) primer extension product (P3.1 -Ext and P1 .1-Ext).
  • FIG. 61 schematically shows certain embodiments of the localization of probe segments which can bind predominantly complementary to the fourth (and possibly the second) primer extension product (P4.1 -Ext and P2.1-Ext).
  • Figs. 62-67 schematically show certain embodiments of probes.
  • FIGS. 68-71 schematically show certain embodiments of the localization of a target sequence 1-3 in the starting nucleic acid 1.1, an amplification fragment 1.1 (P1 .1-Ext and P2.1-ext), and the second amplification fragment 2.1 (P3. 1-Ext and P4.1 -xt).
  • a method for amplification comprises the steps:
  • first primer oligonucleotide hybridizing a first primer oligonucleotide to the 3 'segment of a template nucleic acid fragment (template strand, starting nucleic acid chain) of a first nucleic acid to be amplified which comprises a target sequence
  • first primer oligonucleotide comprises the following regions: a first region capable of binding sequence-specifically to the 3 'segment of a template strand of the first nucleic acid to be amplified and being complementary to at least a portion of the target sequence;
  • a second region which adjoins the 5 'end of the first region or is linked via a linker, which second region can be bound by a controller oligonucleotide and by a polymerase used for the first amplification under the selected reaction conditions in the Essentially remains uncopied;
  • a second region that is substantially complementary or fully complementary to the first region of the first primer oligonucleotide and / or the first primer extension product
  • a third region which is substantially complementary or fully complementary at least in part to the polymerase-synthesized portion of the first primer extension product
  • controller oligonucleotide does not serve as a template for primer extension of the first primer oligonucleotide
  • the controller oligonucleotide comprises a sequence segment which is identical or substantially identical to a strand of the template strand (the target sequence), and
  • the controller oligonucleotide binds to the first region of the first primer extension product
  • the controller oligonucleotide binds to the second region of the first primer extension product and at least partially to the synthesized region of the first primer extension product displacing complementary portions of the template strand of the first nucleic acid to be amplified; wherein the polymerase synthesized region of the first primer extension product which is the second primer Oligonucleotide complementary segment of the first primer extension product is single-stranded under the reaction conditions;
  • Extension product (obtained in step 2.D, P4.1 -ext part 1), wherein the third primer oligonucleotide comprises a first region substantially sequence-specific to a segment of the fourth primer extension product (P4.1-Ext part 1) can bind; 2.G) extension of the third primer oligonucleotide by a second polymerase using the fourth primer extension product (P4.1-Ext Part 1) as a template to give a complete third primer extension product (P3.1-Ext), the third primer extension product (P3.1 -Ext) and the fourth primer
  • Extension product (P4.1 -Ext part 1) are present as a double strand
  • step 2B P3.1 -ext part 1
  • the fourth primer oligonucleotide comprises a first region capable of substantially sequence-specific binding to the polymerase-synthesized region of the third primer extension product
  • step 2I, P4.1 -Ext hybridizing a third primer oligonucleotide to the complete fourth primer extension product (obtained in step 2I, P4.1 -Ext), wherein the third primer oligonucleotide comprises a first region substantially sequence-specific to a segment of the fourth Primer extension product (P4.1-Ext);
  • this aspect of the invention may also be formulated as a method of amplifying a nucleic acid (Figs. 1, 5):
  • first amplification for the amplification of first amplification fragments 1.1 comprising a target sequence
  • first amplification comprises the following steps: a) hybridization of a first primer oligonucleotide to the 3 'segment of a strand of a nucleic acid to be amplified, the nucleic acid chain to be amplified comprises a target sequence,
  • first primer oligonucleotide comprises the following ranges:
  • a first primer region in the 3 ' segment of the first primer oligonucleotide which can bind sequence-specifically to a strand of a nucleic acid chain to be amplified
  • controller oligonucleotide c) binding of the controller oligonucleotide to the polynucleotide tail of the second region of the first extended primer oligonucleotide, wherein the controller oligonucleotide comprises the following regions:
  • a second single-stranded region which is substantially complementary to the first region of the first primer oligonucleotide and can bind to it
  • a third single-stranded region which is substantially complementary to at least one segment of the polymerase-synthesized extension product of the first primer extension product
  • controller oligonucleotide does not serve as a template for primer extension of the first primer oligonucleotide
  • extension of the second oligonucleotide primer with polymerase to form a second primer extension product, wherein the extension is up to and including the first primer region of the first primer oligonucleotide and this first primer region is copied from the polymerase, wherein the polynucleotide Tail of the second region remains uncopied; h) repeating steps a) -g) until the desired level of amplification is achieved.
  • a second amplification which is carried out by using, in the first amplification reaction, first amplification fragments 1 .1 as templates and using a third primer and a fourth primer and a second polymerase, and suitable cofactors for propagating a second amplification Fragments 2.1 a target sequence or at least a segment of the target sequence (or its complementary sequence), where
  • the third primer oligonucleotide can bind to the second primer extension product and / or to the fourth primer extension product in a substantially sequence-specific manner and can be extended by the second polymerase,
  • the fourth primer oligonucleotide can bind to the first primer extension product and / or to the third primer extension product in a substantially sequence-specific manner and can be extended by the second polymerase,
  • the resulting primer extension products starting from the third and fourth primers can serve as templates for an exponential amplification of the second amplification fragments 2.1.
  • a method according to one of the above-mentioned aspects may be characterized in that the second amplification is a PCR (polymerase chain reaction, polymerase chain reaction, also called PCR amplification) and is carried out under such conditions as an amplification of second amplification fragments 2.1 comprehensively allows a target sequence.
  • PCR polymerase chain reaction, polymerase chain reaction, also called PCR amplification
  • a method according to one of the above-mentioned aspects may be characterized in that at least the second amplification is carried out by PCR amplification in the presence of a detection system comprising an oligonucleotide probe.
  • a method according to any of the above aspects may be characterized in that the second amplification is a PCR amplification and is carried out under conditions which include at least one step in which primers are hybridized and / or extended by polymerase, and at least a step in which the resulting primer extension products are converted into single-stranded form (denatured).
  • a method according to one of the above-mentioned aspects may be characterized in that the second amplification is a PCR amplification and is carried out until the desired amount of amplification fragments 2.1 are synthesized.
  • a method according to one of the above-mentioned aspects may be characterized in that the third primer oligonucleotide of the second amplification in the 3 ' segment of the second primer extension product and / or in the 3 ' segment of the fourth primer
  • Extension product and can be extended by a polymerase.
  • a method according to one of the above-mentioned aspects may be characterized in that the fourth primer oligonucleotide of the second amplification in the 3 ' segment of the first primer extension product and / or in the 3 ' segment of the third primer
  • Extension product and can be extended by a polymerase. Furthermore, a method according to one of the above-mentioned aspects may be characterized in that the third primer oligonucleotide of the second amplification binds with its 3 ' segment to the second primer extension product and / or the fourth primer extension product and is extended by a polymerase can.
  • a method according to one of the above-mentioned aspects may be characterized in that the fourth primer oligonucleotide of the second amplification binds with its 3 ' segment to the first primer extension product and / or the third primer extension product and is extended by a polymerase can.
  • a method according to one of the above-mentioned aspects may be characterized in that the extension product of the third primer oligonucleotide and / or the extension product of the fourth primer oligonucleotide of the second amplification comprises at least one segment of the target sequence or its complementary sequence.
  • the third single-stranded region of the controller oligonucleotide is substantially complementary to the segment of the polymerase-synthesized extension product of the first primer extension product that immediately adjoins the first primer region.
  • a method according to one of the above-mentioned aspects may be characterized in that the displacement of the strand complementary to the first primer extension product of the nucleic acid to be amplified takes place until this complementary strand of the nucleic acid to be amplified is detached from the first primer extension product.
  • step (f) of the method is modified to include hybridizing a second oligonucleotide primer to the first primer extension product, wherein at least a partial displacement of the controller -Oligonukleotides from the bond with the first extension product by strand displacement occurs.
  • step (g) of the method is modified to include displacement of the controller oligonucleotide from binding with the first primer extension product with the participation of the polymerase.
  • a method according to any of the above aspects may be characterized in that step (h) of the method is modified to include binding of the controller oligonucleotide to the uncopied polynucleotide tail of the first extended primer oligonucleotide and displacement of the second Primer extension product from binding to the first primer extension product, with simultaneous formation of a complementary duplex with a segment of the first specific extension product of the first primer oligonucleotide.
  • a method according to any one of the above aspects may be characterized in that the repetition of the steps is carried out under conditions which allow the repetition of steps (a) to (g).
  • a method according to any one of the above aspects may be characterized by comprising simultaneously amplifying the first and second primer extension products in an exponential reaction using the first and second primer oligonucleotides and the controller oligonucleotide, wherein the formed primers Extension products act as templates for mutual synthesis.
  • the yield of individual products and intermediates depends on several factors. For example, higher concentrations of individual primers generally favor the yield of products / intermediates. Furthermore, the formation of products / intermediates can be influenced by the choice of the reaction temperature and the binding affinity of individual reactants to each other (affinity of binding of individual primers to their complementary primer binding sites within template strands): in general, longer oligonucleotides, for example, bind better at higher temperatures as shorter oligonucleotides, CG content may also play a role in complementary sequence segments: CG-richer sequences also bind stronger at higher temperatures than AT-rich sequences. Furthermore, modifications such as MGB or 2-amino-dA or LNA can increase the binding strength of primers to their respective complementary segments, which also results in preferential binding of primers at higher temperatures.
  • Hybridizing a first primer oligonucleotide (P1.1) to a nucleic acid to be amplified with a target sequence (either starting nucleic acid 1 .1 or P2.1-Ext), wherein the first primer oligonucleotide (P1.1) comprises the following ranges:
  • a first region which can bind sequence-specifically to a region of the nucleic acid to be amplified (P2.1 E1),
  • a second region (P1 .1.2) which connects to the 5 'end of the first region or is connected via a linker, wherein the second region can be bound by a controller oligonucleotide, and remains essentially uncoated for the polymerase used under the chosen reaction conditions;
  • first primer extension product (P1 .1-Ext) which comprises a synthesized region in addition to the first primer oligonucleotide (P1.1) (P1.1 E1 to P1 .1 E4), which is substantially complementary to the nucleic acid to be amplified or to the target sequence, wherein the first primer extension product and the nucleic acid to be amplified are present as a double strand;
  • Binding of a controller oligonucleotide (C1 .2) to the first primer extension product (P1.1 -Ext), wherein the controller oligonucleotide (C1.2) comprises the following ranges:
  • a second region (C1.2.2) which is complementary to the first region of the first primer oligonucleotide (P1.1 .1), and
  • controller oligonucleotide (C 1 .1) does not serve as a template for a primer extension of the first primer oligonucleotide (P1 .1), and the controller oligonucleotide (C 1,1) to the first primer extension product (P1.1 E6, P1.1 E5, P1.1 E4) while displacing the region (P2.1 E1, P2.1 E2) which is complementary to this region, of the nucleic acid to be amplified;
  • the invention comprises the following aspects: A method according to aspect 1, wherein the nucleic acid fragment comprising a target sequence is provided in single-stranded form.
  • nucleic acid fragment comprising a target sequence in double stranded form comprising a nucleic acid fragment including a first target sequence, the start nucleic acid chain, and a complementary nucleic acid fragment
  • this double stranded nucleic acid fragment is converted to single stranded form prior to the first amplification.
  • temperatures which allow a predominantly complementary binding of primers to complementary sequence segments of the synthesized strands and a primer extension by the second polymerase.
  • temperature includes ranges from 20 ° C to about 80 ° C.
  • the first primer oligonucleotide comprises in its first region a sequence segment which is capable of complementary binding to the first target sequence in its 3 ' segment.
  • the second primer oligonucleotide comprises in its 3 ' region a sequence segment which is substantially identical to a portion of the first target sequence, said portion being in the 5 ' segment of the target sequence
  • controller oligonucleotide comprises in its second and third regions a sequence segment which is substantially identical to a portion of the target sequence.
  • the third primer oligonucleotide comprises in its 3 ' region a sequence segment which is capable of substantially complementary binding to the second primer extension product of the first amplification fragment 1.1.
  • the third primer oligonucleotide comprises in its 3 ' region a sequence segment which is capable of substantially complementary binding to the second primer extension product of the first amplification fragment 1.1, and further includes that 3 ' region first segment complementary segment.
  • the fourth primer oligonucleotide comprises in its 3 ' region a sequence segment which is capable of substantially complementary binding to the first primer extension product of the first amplification fragment 1.1.
  • the fourth primer oligonucleotide comprises in its 3 ' region a sequence segment which is capable of substantially complementary binding to the first primer extension product of the first amplification fragment 1.1 and further comprising a segment of that 3 ' region which is substantially identical to a portion of the first target sequence, this portion being in the 5 ' region of the target sequence
  • Primer extension products (P4.1 -Ext part 1) and (P4.1 -Ext) are identical.
  • Primer extension products (P4.1-Ext Part 1) and (P4.1 -Ext) are identical, but differ the primer extension product of the second and fourth primer oligonucleotide.
  • Primer extension products (P3.1 -Ext part 1) and (P3.1 -Ext) are identical.
  • the nucleic acid to be amplified in the first amplification comprises a first target sequence.
  • at least one start nucleic acid chain 1.1 is added to the reaction which comprises a target sequence and can serve as template for the synthesis of the first amplification fragment.
  • the synthesized primer extension products of the first amplification (the first amplification fragment 1 .1) comprise the target sequence.
  • This first amplification fragment 1 .1 can serve as start nucleic acid chain 2.1 with a template function for the second amplification.
  • the primer extension products synthesized in the second amplification (P3.1-Ext and P4.1-Ext) (the second amplification fragment 2.1) comprise at least portions of the first target sequence.
  • substantially complementary in the context of the present disclosure means that two nucleic acids, in particular their mutually complementary regions, have not more than 5, 4, 3, 2, or 1 mismatches to one another.
  • the steps of the first amplification are repeated at least twice.
  • the second amplification is based on a copy number of the first amplification fragment (1.1). is started, which is present in the range of 10 to 1 E12, in particular from 100 to 1 E10, in particular from 10E3 to 10E9.
  • the steps of the second amplification are repeated until the amplification fragment (2.1) is present in a copy number in the range from 10E5 to 1E16, in particular from 10E5 to 1E14, in particular 10E6 to 1 E14.
  • the steps of the second amplification are repeated at least twice.
  • the detection system comprises at least one
  • Oligonucleotide probe labeled with a fluorescent reporter Oligonucleotide probe labeled with a fluorescent reporter.
  • the detection system comprises at least one
  • Oligonucleotide probe labeled with a fluorescent reporter and a fluorescence quencher.
  • the detection system comprises at least one
  • An oligonucleotide probe labeled with a donor fluorophore capable of forming a FRET pair with the fluorescent reporter is an oligonucleotide probe labeled with a donor fluorophore capable of forming a FRET pair with the fluorescent reporter.
  • an oligonucleotide probe comprises a sequence segment which is substantially complementary to at least one of the primer extension products formed (P1 .1-Ext, P2.1-Ext, P3.1-Ext, P4.1-Ext) and which is at least hybridize to one of the formed primer extension products under suitable conditions (hybridization conditions).
  • this sequence segment is complementary to the first and / or the third primer extension product.
  • this sequence segment is complementary to the first and / or the third primer extension product, wherein the oligonucleotide probe can bind complementary in the 3 ' segment of the respective primer extension product which is not complementarily bound by the controller oligonucleotide.
  • this sequence segment is complementary to the second and / or the fourth primer extension product.
  • this sequence segment of the oligonucleotide probe comprises a length which is in the range between 10 nucleotides and 50, in particular between 15 and 40, in particular between 15 and 30 nucleotides.
  • this sequence segment of the oligonucleotide probe does not comprise a sequence region that is substantially complementary to the controller oligonucleotide. In a further embodiment, this sequence segment of the oligonucleotide probe comprises a sequence region which is substantially complementary to the controller oligonucleotide, the length of this segment being less than 20 nucleotides, in particular less than 15 nucleotides, in particular less than 10 nucleotides.
  • this sequence segment of the oligonucleotide probe does not comprise a sequence region that is substantially complementary to one of the primer oligonucleotides.
  • this sequence segment of the oligonucleotide probe comprises a sequence region which is substantially complementary to one of the primer oligonucleotides, the length of this segment being less than 20 nucleotides, in particular comprising less than 15 nucleotides, in particular less than 10 nucleotides, in particular less than 5 nucleotides.
  • this sequence segment of the oligonucleotide probe does not comprise a sequence region that is substantially identical to the sequence of the third region of the controller oligonucleotide.
  • this sequence segment of the oligonucleotide probe comprises a sequence region which is substantially identical to the sequence of the third region of the controller oligonucleotide, the length of this segment being less than 20 nucleotides, in particular less than 15 nucleotides, in particular less than 10 nucleotides.
  • the controller oligonucleotide comprises one of the following components (a fluorescence reporter and / or a fluorescence quencher and / or a donor fluorophore), wherein at least one of these components is located in the third region of the controller oligonucleotide.
  • the oligonucleotide probe is at least partially cleavable by a 5 ' -3 ' nuclease.
  • an oligonucleotide probe comprises a sequence segment which is substantially complementary to the target sequence or its portion encompassed by one of the amplification products, the length of this segment being in the range of between 5 and 50 nucleotides, in particular between 10 and 40, in particular between 15 and 30 nucleotides.
  • an oligonucleotide probe does not comprise a sequence segment substantially complementary to the target sequence or its complementary strand.
  • an oligonucleotide probe comprises a sequence segment not substantially complementary to the target sequence or its complementary strand.
  • the binding of the oligonucleotide probe to the respective complementary segment of the primer extension product formed under suitable hybridization conditions leads to formation of a double strand.
  • the hybridization conditions are present during the process.
  • the reaction is illuminated with light of a suitable wavelength for the generation of a fluorescence signal and the detection of the fluorescence signal from the fluorescence reporter, whereby the intensity of the fluorescence signal is measured.
  • the detection of the fluorescence signal is carried out with suitable optical / physical means, in which either the increase of the fluorescence signal or the decrease of the fluorescence signal is measured.
  • suitable sensors By means of suitable sensors, the intensity of the fluorescence signal can be converted into measured values which, after appropriate calibration, make it possible to determine whether a desired target sequence is present in the reaction mixture or not.
  • reporter-quencher pair An essential aspect of the fluorescence detection system is the reporter-quencher pair.
  • the quencher When the quencher is in close proximity to the reporter, no signal is emitted upon exposure to the excitation light.
  • reporter and quencher molecules are held in close proximity due to a complementary star sequence, no fluorescence signal appears even under illumination.
  • reporters and quenchers are placed in a spatial distance that causes the quencher to be so far from the reporter molecule that the reporter molecule will emits fluorescence radiation when the reaction mixture is irradiated with an excitation light source. The distance between reporter and quencher depends on the molecules used.
  • the emission of light after irradiation of the fluorescence reporter is then reduced or almost completely reduced when the quencher and reporter are at a distance of less than 25 nucleotides from each other.
  • This removal can be effected either by the nucleotide sequence or by special spacing molecules, such as linkers or spacers.
  • the fluorescent reporter can form a specific reporter-donor (FRET) pair with a donor fluorophore capable of transmitting the absorbed energy to fluorescent reporters by fluorescence resonance energy transfer (FRET) ,
  • FRET fluorescence resonance energy transfer
  • a reporter-donor pair comprising a fluorescent reporter and a matching donor fluorophore and forming a fluorescence resonance energy transfer pair may be designed so that only one of the partners of such a FRET pair is coupled to the oligonucleotide probe and the other partner is coupled to the controller oligonucleotide.
  • Both fluorescent reporter and donor fluorophore are derived from the respective oligonucleotide coupled that in the non-hybridized state of the oligonucleotide probe, the donor fluorophore is unable to transfer the absorbed energy to the fluorescent reporter.
  • the controller oligonucleotide is a component of the detection system and comprises a fluorescent reporter and / or a fluorescence quencher and / or a donor fluorophore.
  • a fluorescent reporter and / or a fluorescence quencher and / or a donor fluorophore By simultaneous hybridization of the controller oligonucleotide to the synthesized first or third primer extension product and the oligonucleotide probe to the 3 ' segment of the same first and / or third primer extension product, binding is made to adjacent sequence positions of the first and / or third primer Extension product, resulting in spatial proximity of the donor fluorophore and the fluorescence reporter. This reduces the distance between the fluorescent reporter and donor fluorophore so much that FRET can take place from donor fluorophore to fluorescent reporter. This leads to the generation of a fluorescence signal of the fluorescence reporter and results in a detectable increase in the fluorescence signal of the fluorescence reporter.
  • the distance between the donor fluorophore and the fluorescent reporter after hybridization should be less than 25, in particular less than 15 and in particular less than 5 nucleotides.
  • the wavelength of the light upon excitation is absorbed by the donor fluorophore and transferred to the reporter, thereby emitting light that can be detected.
  • the following embodiments are preferred, wherein either the fluorescence reporter or the fluorescence quencher or the donor fluorophore are coupled either to the controller oligonucleotide, in particular in the third region of the controller oligonucleotide:
  • the coupling to the controller oligonucleotide preferably takes place in the third region of the controller oligonucleotide
  • the coupling to the controller oligonucleotide preferably takes place at the 5 ' end or the third region or in its vicinity, for example 2 to about 10 nucleotides from the 5 ' end of the third region
  • Coupling to the oligonucleotide probe is preferably carried out in the middle region of the oligonucleotide probe,
  • - Coupling to the oligonucleotide probe is preferably carried out in the 3 ' segment of the oligonucleotide probe.
  • the distance between both elements of a hybridized FRET pair of oligonucleotides hybridized to the first primer extension product is less than about 30 nucleotides.
  • the oligonucleotide probe comprising a complementary 3 ' segment to one of the primer extension products is so modified that the polymerase is unable to extend that 3 ' end.
  • the oligonucleotide probe comprising a complementary 3 ' segment to one of the primer extension products so that the polymerase is able to extend that 3 ' end, the oligonucleotide probe serving as a PCR primer.
  • a method for detecting amplification wherein two or more nucleic acid chains are amplified in which a specific detection system is used for each nucleic acid chain to be amplified.
  • Method for detecting amplification wherein two or more nucleic acid chains to be amplified are amplified, in which the detection of the amplification of at least two nucleic acid chains to be amplified is effected by a uniform detection system.
  • the amplification comprises an asymmetric amplification of one of the primer extension products.
  • the excitation of the fluorescence reporter and the measurement of the fluorescence signal of the fluorescence reporter takes place during the amplification.
  • the excitation of the donor fluorophore and the measurement of the fluorescence signal of the fluorescence reporter takes place during the amplification.
  • the excitation of the fluorescence reporter and the measurement of the fluorescence signal of the fluorescence reporter takes place after the amplification.
  • the oligonucleotide probe is a DNA oligonucleotide.
  • the oligonucleotide probe is a DNA oligonucleotide and the 3 ' end is blocked so that the polymerase can not extend it.
  • the oligonucleotide probe comprises a complementary sequence segment to the first and / or the third primer extension product, this sequence segment comprising a length of 8 to about 60 nucleotides.
  • An oligonucleotide probe comprises a complementary sequence segment to the 3 ' segment of the first and / or third primer extension product which is not bound by the controller oligonucleotide, this segment being from 8 to about 40 nucleotides in length.
  • An oligonucleotide probe comprising at least one further Modifkation selected from the following group: linker (such as HEG, C3, C6), phosphate-sugar backbone modifications (such as PTO, 2 '-0-Me, RNA, PNA, LNA modifications) ,
  • the third primer oligonucleotide and / or the fourth primer oligonucleotide have a second region which adjoins the 5 'end of the first region or is linked via a linker, the second Area can not bind complementary to the first primer extension product or to the second primer extension product.
  • the second region of the third and / or fourth primer oligonucleotide lies in the 5 ' direction from the first region of the third and / or fourth primer oligonucleotide.
  • the third primer oligonucleotide and / or the fourth primer oligonucleotide comprises a barcode sequence.
  • the separation of the double strand from the first primer extension product and the fourth primer extension product and the double strand from the second primer extension product and the third primer extension product is carried out by thermal denaturation, in particular in a Temperature in the range of 85 ° C to 105 ° C.
  • the first polymerase is capable of strand displacement during synthesis.
  • the second polymerase is a thermostable polymerase.
  • the first polymerase and the second polymerase are identical.
  • the first primer oligonucleotide and the third primer oligonucleotide are substantially identical.
  • the second primer oligonucleotide (P2.1) and the third primer oligonucleotide (P4.2) are substantially identical.
  • substantially identical in the context of the invention means in particular that two nucleic acid sequences with not more than 5, 4, 3, 2, or 1 Mismatches to each other.
  • the reaction conditions of the first amplification are selected such that no spontaneous dissociation of the first amplification product can occur.
  • the second amplification is carried out at at least two temperatures, wherein at a first temperature the hybridization and extension of the third and / or fourth primer oligonucleotide to the first and / or second and / or third and third or fourth primer extension product, and at a second temperature separation of the resulting duplex.
  • the second amplification is carried out at at least three temperatures, wherein at a first temperature, the hybridization of the third and / or fourth primer oligonucleotide to the first and / or second and / or third and / or fourth primer extension product takes place, at a second temperature the extension of the third and / or fourth primer, and at a third temperature the separation of the resulting double strands.
  • the first amplification and the second amplification are carried out in the same reaction batch.
  • the second polymerase, the third primer oligonucleotide and / or the fourth primer oligonucleotide are activatable, and / or the controller oligonucleotide is deactivatable.
  • Activatable polymerases may be, for example, reversible inactivated polymerases such as thermostable polymerase (hot-start polymerase) or polymerases which are reversibly inactivated by an antibody or a chemical modification.
  • reversible inactivated polymerases such as thermostable polymerase (hot-start polymerase) or polymerases which are reversibly inactivated by an antibody or a chemical modification.
  • Activatable primers may be reversible inactivated primer, for example oligonucleotides with a protected 3 'OH group, wherein the protecting group in particular by a polymerase with 5' - exonuclease activity is removable. It would be advantageous in the first amplification to use a polymerase without 5 ' exonuclease activity.
  • the controller oligonucleotide can be inactivated or cleaved, for example, by a polymerase with 5 ' exonuclease activity.
  • the controller oligonucleotide may also be cleaved and thereby removed by other enzymatic or chemical methods prior to the second amplification.
  • the first polymerase is inactivated before carrying out the second amplification. This can be achieved, for example, by thermal denaturation, when the first polymerase is a non-thermostable polymerase.
  • the first amplification is carried out in a first reaction batch and the second amplification in a second reaction batch.
  • an aliquot of the first reaction mixture is added to the second reaction mixture.
  • the complete first reaction batch is introduced into the second reaction mixture.
  • the third portion of the controller oligonucleotide is substantially complementary to the portion of the synthesized portion of the first primer extension product that immediately adjoins the primer oligonucleotide portion of the first primer extension product.
  • this improves the sequence-specific displacement of the nucleic acid to be amplified.
  • the controller oligonucleotide comprises a fourth region capable of substantially sequence-specific binding to the 3 ' region of the third primer oligonucleotide.
  • the fourth region can lie within the second region and / or the third region of the controller oligonucleotide or include sequence segments of the second and / or third region, wherein the fourth region in particular has a length of 9 to 30 nucleotides, in particular 10 to 20 Nucleotides, or 12 to 16 nucleotides.
  • substantially sequence-specific in the context of the present disclosure means that the mutually complementary regions of the primer oligonucleotide and the nucleic acid to be amplified have not more than 5, 4, 3, 2, or 1 mismatches.
  • the controller oligonucleotide comprises one or more nucleotide modifications designed to prevent the extension of a primer bound to the controller oligonucleotide.
  • nucleotide modifications designed to prevent the extension of a primer bound to the controller oligonucleotide.
  • several 2 '-0-alkyl modification can block or prevent such an extension.
  • the controller oligonucleotide comprises at least 5 such modifications, more preferably at least 10 modifications.
  • the controller oligonucleotide consists entirely of nucleotide modifications.
  • the portion of the polymerase-synthesized portion of the first primer extension product that is substantially complementary to the third portion of the controller nucleotide has a length in the range of 5 nucleotides to 70 nucleotides.
  • the third single-stranded region of the controller oligonucleotide is fully complementary to the 5 ' segment of the first primer extension product, the length of this complementary sequence segment being of at least 3 to 70 nucleotides, or of at least 5 to 50 nucleotides , in particular embodiments of 5 to 40 nucleotides, of 5 to 30 nucleotides, in particular of 5 to 20 nucleotides.
  • the first amplification is carried out essentially isothermally.
  • the second amplification is carried out at three temperatures, wherein at the first temperature the hybridization of the third or fourth primer oligonucleotide and optionally the initial extension of the primer is performed, and at a second temperature the complete Extension of the primers and at a third temperature the separation of formed primer extension products from their respective template strands.
  • the first temperature is in the range of 25 ° C to 65 ° C
  • the second temperature in the range of 65 ° C to 80 ° C
  • the third temperature in the range of 85 ° C to 105 ° C.
  • the nucleic acid to be amplified has a length in the range from 20 nucleotides to 300 nucleotides.
  • the first primer oligonucleotide has a length in the range of 15 nucleotides to 100 nucleotides.
  • the second primer oligonucleotide has a length in the range of 15 nucleotides to 100 nucleotides.
  • the controller oligonucleotide has a length in the range from 20 nucleotides to 100 nucleotides.
  • the third primer oligonucleotide has a length in the range of 15 nucleotides to 60 nucleotides.
  • the fourth primer oligonucleotide has a length in the range of 15 nucleotides to 60 nucleotides.
  • the third primer oligonucleotide has at least one nuclease-resistant nucleotide modification, for example, a PTO (phosphorothioate) or 2 '-0-alkyl modification.
  • the fourth primer oligonucleotide has at least one nuclease-resistant nucleotide modification, for example, a PTO or 2 '-0-alkyl modification.
  • the first primer oligonucleotide in the second region is a modification or several modifications in particular immediately after the first region of the first primer oligonucleotide which stops / prevents the polymerase used from copying the second region.
  • the first primer oligonucleotide in the second region is a modification or several modifications in particular immediately after the first region of the first primer oligonucleotide which stops / prevents the polymerase used from copying the second region.
  • only the first region of the first primer oligonucleotide is thereby copied.
  • nucleotide modifications which, while capable of complementary binding to the first primer extension product, are not accepted by the polymerase as a template.
  • nucleotide modifications set nucleotide compounds with modified phosphate-sugar backbone moieties, for example, 2 '-0-alkyl-RNA modifications (for example, 2 - OMe), LNA modifications or morpholino Modifkationen is generally prevents the. Presence of such modifications in one strand of a DNA-dependent polymerase upon reading such a strand. The number of such modifications may be different, usually few modifications (between 1 and 20) may be sufficient to prevent a polymerase from reading such a strand.
  • nucleotide modifications can be used, for example, at or about the binding site of the first primer oligonucleotide to the controller oligonucleotide and / or as constituents of the second region of the first primer oligonucleotide.
  • kit for carrying out the method according to the invention according to the aspects described above or their specific embodiments or alternatives.
  • the kit according to the invention comprises:
  • a first primer oligonucleotide having the following ranges:
  • a second region connected to the 5 'end of the first region or linked via a linker, wherein the second region can be bound by a first controller oligonucleotide and one for the Amplification used polymerase remains essentially unkopiert under the selected reaction conditions;
  • first primer oligonucleotide can be extended by a polymerase to a first primer extension product comprising a synthesized region in addition to the first primer oligonucleotide;
  • the second primer oligonucleotide comprises a region capable of sequence-specific binding to the synthesized region of the first primer extension product and can be extended from a polymerase to a second primer extension product which is adjacent to the second primer Oligonucleotide comprises a synthesized region
  • controller oligonucleotide comprising the following ranges:
  • a second region which is substantially complementary or complete to the first region of the first primer oligonucleotide
  • a third region that is substantially complementary or fully complementary to at least a portion of the synthesized region of the first primer extension product
  • controller oligonucleotide does not serve as a template for a primer extension of the first primer oligonucleotide, and the controller oligonucleotide is attached to the primer
  • Extension product can bind under displacement of the complementary region of the second primer extension product
  • a third primer oligonucleotide wherein the third primer oligonucleotide comprises a first region capable of sequence-specific binding to a segment of the second primer extension product and can be extended from a polymerase to a third primer extension product (P3.1-Ext) which comprises a synthesized region adjacent to the third primer oligonucleotide; and
  • a fourth primer oligonucleotide wherein the fourth primer oligonucleotide comprises a first region capable of sequence-specific binding to the synthesized region of the first primer extension product, and extendable from a polymerase to a fourth primer extension product which is adjacent to the fourth primer - Oligonucleotide comprises a synthesized region.
  • the kit further comprises at least one polymerase.
  • the kit comprises a first polymerase designed to extend the first and second primer oligonucleotides, and a second polymerase designed to extend the third and fourth primer oligonucleotides.
  • the first polymerase has no 5 ' exonuclease activity and the second polymerase has no 5 ' exonuclease activity.
  • the second polymerase may be a thermostable polymerase.
  • the second polymerase, the third primer oligonucleotide and / or the fourth primer oligonucleotide is activatable, and / or the controller oligonucleotide is deactivatable.
  • kit according to the above aspect for carrying out the method according to the invention according to the above-described aspects or their specific embodiments or alternatives is provided.
  • the combination according to the invention comprises two amplification processes to be carried out in succession.
  • the first partial amplification the first amplification procedure
  • nucleic acid chains comprising a target sequence are amplified.
  • This second partial amplification will in many cases be a conventional PCR.
  • the amplification proceeds from the target nucleic acids or nucleic acids comprising the target nucleic acids which were amplified in the first partial amplification.
  • PCR fragments are amplified.
  • these PCR fragments comprise target sequences or portions of target sequences.
  • target sequences can be flanked by using barcoding primers or detection-specific primers.
  • detection can be detected using PCR probes (e.g., so-called Taqman probes).
  • immobilization of target sequences to the solid phase using immobilized primers can be used during the PCR phase so that the PCR proceeds as solid-phase PCR.
  • the first amplification method according to the invention is intended to be able to synthesize or amplify nucleic acid chains having a defined sequence composition, it being possible for generated products to serve as starting nucleic acid chains with template function for subsequent PCR, and the PCR amplification being carried out using at least one own PCR primer ,
  • An object of the invention is further achieved by providing a first amplification method (a first partial amplification) and corresponding means for carrying it out.
  • a first amplification method (a first partial amplification) and corresponding means for carrying it out.
  • the implementation of this first amplification method has already been described in PCT application PCT / EP2017 / 07101 1 and European application 16185624.0 described.
  • PCT / EP2017 / 07101 1 and European application 16185624.0 described.
  • the skilled person is referred to this application.
  • the second amplification method (the second partial amplification) can be carried out in particular as PCR, wherein at least one of the primers used has a different composition and / or structure than the primers used in the first amplification method. This results in an increase of at least one nucleic acid chain comprising the specific target sequence or its parts.
  • PCR amplification per se is normally capable of generating well-characterized amplification products from the initial template, more specifically in real-time mode, starting from an amount greater than about 1000 copies, more preferably greater than about 100,000 copies Generate signals.
  • the PCR must include more cycles of synthesis, which can increasingly lead to defective amplification products or signals. This is especially the case if the PCR has to amplify a sequence variant (eg a mutation or allelic variant of a sequence) which is present in small amounts in the batch and the PCR amplification in the presence of a large amount of another sequence variant (eg. Sequence or allelic variant).
  • amplification-dependent detection methods can benefit from further enhancement of the specificity of amplification techniques, e.g. in the field of liquid biopsy. Especially in clinical diagnostics there is a need to improve the specificity of measurement methods.
  • the combination of a preceding highly specific amplification method with downstream PCR will be described.
  • the combination makes it possible to increase the initial copy number of nucleic acid chains to be amplified under highly selective amplification conditions of a first amplification method up to the desired amounts, which are then subsequently further amplified under less specific conditions of a PCR amplification.
  • the first amplification method thus has a function of the first amplifier with high specificity.
  • the amount of synthesized copies in the first amplification process will range from about 10 copies to about 1 E 1 1 copies, more preferably from about 100 copies to about 1 .0E 10 copies, preferably from about 1000 to about 10E8 copies.
  • the amount of a target sequence is increased by the first amplification.
  • This increase (based on the starting amount of the target sequence in the batch) is, for example, in the following ranges from about 2 times to about 1 E10. more preferably from about 10 times to about 10E9 times, more preferably from about 10 times to about 10E8 times, preferably from about 10 times to about 10E7 times, more preferably from about 100 times to 10E6 times ,
  • the following concentration ranges are achieved: from about 10 amol / l to about 10 nmol / l, more preferably from about 1 fmol / l to about 1 nmol / l, even better from about 10 fmol / l to about 0.1 nmol / l, in particular from about 10 fmol / l to about 10 pmol / l.
  • the second amplification method plays the role of a second amplifier and allows existing PCR-based methods such as probe insertion or bar coding or sequence coding in NGS-library-preparation or solid phase PCR in combination with specific products of the first amplification.
  • the advantages of the combination are, on the one hand, the reduction in the number of PCR cycles which are necessary for carrying out the amplification up to the desired amount of the end product. This reduces the likelihood or extent of synthesis of products by PCR. The synthesis result can thus have higher specificity than the PCR amplification alone.
  • first the first amplification with required components of the first amplification system takes place in the absence of at least one of the essential components of the second amplification system in the same reaction mixture during the first amplification reaction.
  • PCR primers or a thermostable polymerase are added only prior to PCR amplification.
  • the reaction mixture After completion of the first amplification reaction, the reaction mixture is brought into contact with the components of the second amplification system and PCR amplification is carried out.
  • reaction components of a respective amplification system are present in separate reaction mixtures and / or in separate reaction vessels prior to the start of amplification.
  • the first amplification reaction is carried out in one reaction vessel, and then the synthesis result of this reaction is transferred (completely or partially) to the second reaction vessel, and then the second amplification is carried out.
  • reaction components are added sequentially so that first the components of the first amplification system are added and the first amplification reaction is carried out. Subsequently, the components of the second amplification system are supplied, so that the second amplification can take place. In further embodiments, the addition of the components of the second amplification system to the first amplification approach, so that the reaction is substantially the same Reaction vessel is performed. The sequence of both amplification methods is thus predetermined by the time sequence of the addition of individual components.
  • both reactions can be carried out in particular in a closed system.
  • a closed system may comprise, for example, at least two separate reaction vessels.
  • the transfer of the batch from one reaction vessel to the other also takes place in certain embodiments by means of a closed system.
  • Such separation of the two reactions in a closed reaction vessel system is known, e.g. as separate reaction vessels, which are combined to form a cartridge system or array system or microfluidic system.
  • the first amplification batch comprising the amplified nucleic acids of the first amplification is used for the second amplification without partitioning of the first batch.
  • the first approach is thus essentially completely fed to the second amplification reaction.
  • An amount of synthesized copies from the first amplification process is supplied thereto to the second amplification system and includes, for example, a range from about 10 copies to about 1 E1 1 copies, more preferably from about 100 copies to about 1 E10 copies, preferably from about 1000 to about 10E9 copies , In this case, the amount of a target sequence is increased by the first amplification.
  • This increase (based on the starting amount of the target sequence in the batch) is for example in the following ranges from about 2 times to about 1 E1 1-fold, better from about 10 times to about 1 000 000 000 times, even better of about 10-fold to about 1,00,000,000-fold, preferably from about 10-fold to about 10,000,000-fold, more preferably from about 100-fold to 1,000,000-fold.
  • concentration ranges can be achieved: from about 10 amol / l to about 10 nmol / l, more preferably from about 1 fmol / l to about 1 nmol / l, even better from about 10 fmol / l to about 0 , 1 nmol / l, preferably from about 10 fmol / l to about 10 pmol / l.
  • the second amplification reaction it is preferred to use only a part of the first amplification mixture (an aliquot or a subset) in the second amplification reaction. Due to an amplification by means of the first amplification system, an increase in the amount of nucleic acid chains comprising at least one target sequence takes place. In the second amplification reaction, a portion of this amount may be used as the starting material. This fraction can be added or added to the second reaction mixture, for example. The proportion may for example be relatively low and depends essentially on the requirements of the desired application. Due to the proliferating effect of the PCR, the second amplification can in particular start from more than about 1000 copies of the nucleic acid chains which were amplified sequence-specifically in the first amplification method.
  • the PCR amplification can thus be started when a sufficient amount of the amplified nucleic acid chains has been synthesized in the first amplification process.
  • this amount of synthesized nucleic acid chains may range from, for example, a range of about 10,000 copies to about 1 E11 copies, more preferably from about 10,000 copies to about 1 E10 copies, more preferably from about 10,000 to about 1 E9 copies.
  • the amount of a target sequence is increased as desired by this first amplification.
  • This increase (based on the starting amount of the target sequence in the batch) can be measured as N times the starting amount and is for example in the following ranges from about 2 times to about 1 E1 1-fold, more preferably from about 10 times to about 1 E10-fold, more preferably from about 10 times to about 1 E9-fold, especially from about 10-fold to about 1 E8-fold, especially from about 100-fold to 1 E6-fold.
  • the following concentration ranges are achieved: from about 10 amol / l to about 10 nmol / l, more preferably from about 1 fmol / l to about 1 nmol / l, even better from about 10 fmol / l to about 0.1 nmol / l, in particular from about 10 fmol / l to about 10 pmol / l.
  • a fraction (or aliquot) of this amount can be used for the second amplification reaction.
  • the PCR may start from an amount which includes the following ranges, for example, a range of about 100 copies to about 1 E 1 1 copies, more preferably from about 1,000 copies to about 1 E 10 copies, more preferably from about 1, 000 to about 1 E9 copies. This amount can be considered as a subset of the synthesized nucleic acid sequence containing the target sequence, augmented by the first amplification.
  • the concentration ranges include: from about 10 amol / l to about 100 nmol / l, more preferably from about 1 fmol / l to about 10 nmol / l, more preferably from about 10 fmol / l to about 1 nmol / l, more preferably from about 10 fmol / l to about 10 pmol / l.
  • a PCR amplification can be started starting from these concentrations of the synthesis products of the first amplification.
  • the final concentration of PCR fragments concentration ranges from 0.01 nmol / l up to 5 pmol / l, more preferably from 1 nmol / l to 1 pmol / l comprises ,
  • the first amplification provides an amount of copies which represents a relative excess relative to the necessary amount of copies required or desired as a starting material for the PCR.
  • a portion of the first reaction mixture can be made, for example, by dilution of the first reaction mixture and transfer of a certain volume fraction which contains synthesized nucleic acid chains in the second reaction.
  • the proportion of the amount of specifically synthesized nucleic acid chains transferred thereby, which can serve as a template for generating PCR fragments, may include the following ranges (calculated on the total amount of synthesized products in the first amplification step): from about 1 E-7% from about 1 E-6% to about 90%, more preferably from about 0.0001% to about 50%, more preferably from about 0.001% to about 50%, or from about 0.01% to about 50% %, in particular 0.1% to about 20%.
  • dilution of a controller oligonucleotide may also have a positive effect on the PCR reaction by having this controller oligonucleotide initially present during the second amplification reaction, but its concentration during the second amplification is reduced so that its binding is sufficiently slowed down to the synthesis products or reaction components or the interaction with complementary sequence segments is reduced or occurs insufficiently and thus its effect on the overall reaction process is reduced by this dilution effect.
  • dilution of the reaction components of the first amplification system may also be performed, generally in parallel with dilution of the specific nucleic acid chain provided.
  • the relative amount of components of the first amplification system which are transferred to the second amplification reaction can be represented as a proportion. This proportion refers to the amount of the components used in the first amplification reaction (100%).
  • the transferred proportion of the components of the first amplification system in the second amplification reaction comprises, for example, the following ranges: from about 1 E-7% to about 50%, more preferably from about 1 E-6% to about 30%, even better from about 0.0001% to about 20%, especially from about 0.001% to about 10%, more preferably from about 0.1% to about 10%.
  • the transferred portion of the first reaction mixture may, for example, relate to the concentrations used or to the volume fractions.
  • the transfer of a microliter (1 pl) of the batch comprising the first amplification system (after completion of amplification) to 49 pl of a batch comprising the second amplification system in a 2% (v / v) dilution or dilution of 1:50 result.
  • Higher dilution levels can be prepared accordingly by means of a dilution series.
  • the concentration of the controller oligonucleotide in the first amplification system can be reduced from about 10 pmol / l at a dilution of 1: 1000 to about 10 nmol / l.
  • the transferred share is thus only 0.01%.
  • the same proportion of the synthesized nucleic acid chain comprising a target sequence can be transferred.
  • an amount of about 10,000,000 copies was amplified, and then the amount of products transferred to the PCR reaction at a dilution of 1: 1000 would yield a subset of about 10,000 copies. This initial amount of copies is usually sufficient for the PCR to perform a stable and sufficiently specific amplification reaction.
  • nucleic acid chains e.g. of wild-type molecules or allelic variants.
  • sequence-specific first amplification the specific amplification of target molecules takes place predominantly.
  • Other nucleic acid chains are not or not significantly amplified.
  • the dilution effect also acts on these potentially interfering with the PCR reaction sequences. For example, this reduces the amount of wild-type sequences from 100,000 to about 100. This can have a positive effect on the specificity of the PCR reaction.
  • both methods are performed in one approach (as a "homogeneous assay").
  • the components of both amplification systems must already be present in one batch at the beginning of the first amplification.
  • the required components for both amplification systems are thus supplied to the reaction mixture before the beginning of the first amplification.
  • first the first amplification with required components of the first amplification system takes place in the presence of at least one of the essential components of the second amplification system in the same reaction mixture during the first amplification reaction.
  • the combination of both amplification systems in a reaction mixture may require adaptation of individual components or concentrations and reaction conditions.
  • the length of the 3 ' segment of the third primer which may be complementary to the controller oligonucleotide, is chosen so that this segment does not prevent strand displacement by the controller oligonucleotide. This is essentially achieved by the stability of the double-stranded complex consisting of a controller oligonucleotide and a 3 ' segment of the third primer being sufficiently low under reaction conditions of strand displacement by the controller oligonucleotide (eg 65 ° C. step during the first amplification) in that the binding of the third primer to the controller oligonucleotide is only temporary and does not prevent strand displacement.
  • the length of the 3 ' segment of the third primer may be affected, for example, by the length of the 3 ' segment of the third primer, which would be able to enter such a complex with the controller oligonucleotide.
  • the length of this complex is between 8 and 20 nucleotides.
  • the 3 ' segment of the third primer may have one or more mismatches to the sequence composition of the controller oligonucleotide in the corresponding segment, resulting in destabilization of that complex.
  • one to three mismatches are positioned in position from -10 to -20 with respect to the 3 ' -terminal nucleotide of the third primer. This does not significantly affect the primer function of the third primer while reducing the stability of the complex with the controller oligonucleotide.
  • controller oligonucleotide of both primers and the polymerase of the second amplification system should not be used as a template. This is particularly important for the third oligonucleotide primer, since it comprises a sequence segment which can bind complementarily to the controller oligonucleotide.
  • the structure of the controller oligonucleotides in the segment of its possible complementary binding to the third oligonucleotide primer can therefore be designed to be comparable to that of the first primer of the first set.
  • primer extension of the third oligonucleotide primer is prevented by the controller oligonucleotide, by using nucleotide modifications, preventing the polymerase from extending a third primer bound to the controller oligonucleotide.
  • nucleotide modifications For example, several 2 '-0-alkyl modifications can convey such a blocking effect.
  • the components of the second amplification system may furthermore be advantageous for the components of the second amplification system to be completely or partially in an "inert” or “non-active” or “reversibly inactivated” or “non-reactive” state during the first partial amplification. Only for carrying out the second amplification should such components be converted into an active, i. functional state are transferred.
  • At least one of these PCR components is in a reversibly inactivated form during the first amplification so that this component does not participate in the first amplification reaction or its participation is insignificant.
  • the first amplification then takes place first an activation of these reversibly inactivated components, so that the inactivation is canceled and the components are now in active form in the second amplification reaction and thus can perform their role in the amplification.
  • reversibly inactivated PCR primers or a reversibly inactivated thermostable polymerase are used.
  • hot-start polymerase a reversibly inactivated thermostable polymerase
  • the activation of components occurs continuously under PCR conditions.
  • reversibly inactivated primers are known to the person skilled in the art (for example termolabile primers so-called CleanAmp Primers Trilink Technologies). Such primers can be activated by heating so that the polymerases can start synthesis from this primer.
  • Other examples of reversibly inactivated primers include primers having a 3 '-terminal nucleotide which comprises a modified sugar radical, for example a blocked 3' -OH group (for example, by a C3-linker at the 3 'position or primers having terminal dideoxy Nucleotides, Sambrook et al NAR 1998 p.3073).
  • such primers have a 3 ' -terminal mismatch to template.
  • a polymerase is used for the first amplification, which has no 3 ' -5 ' exonuclease activity (eg Bst polymerase or its modifications).
  • Bst polymerase eg Bst polymerase or its modifications.
  • the activation is usually carried out by enzymatic cleavage of the 3 ' -terminal nucleotide together with the modified sugar residue.
  • primers in combination with a thermostable polymerase comprising 3 ' - 5 ' exonuclease activity (so-called proofreading polymerases).
  • a thermostable polymerase comprising 3 ' - 5 ' exonuclease activity
  • proofreading polymerases thermostable polymerase comprising 3 ' - 5 ' exonuclease activity
  • a terminal mismatch facilitates the function of the polymerase 3 ' -5 ' exonuclease.
  • such "inactive" primers bind to their complementary positions on templates. After binding to the template, terminal nucleotides including a blocking group can be removed by exonuclease activity (which is associated with polymerase).
  • thermostable polymerases may also be modified also cleave terminal nucleotides with a blocked 3 '-OH group, if this to the template are complementary (for example, Vent or Deep Vent polymerases).
  • such primers can comprise a 3 '-5' nuclease non-cleavable bond, such as one or more phosphorothioate modifications (PTO modifications), for example, in positions “minus 2" or “ minus 3 “or from” minus 2 "to” minus 7 ". This prevents excessive degradation of primers.
  • PTO modifications phosphorothioate modifications
  • the reversibly activated polymerases include, for example, those which are reversibly inactivated by means of an antibody or which are reversibly inactivated by means of a chemical modification (AmpliT aq polymerase).
  • AmpliT aq polymerase Several commercial suppliers have developed such "hot-start" polymerases for PCR (Qiagen, Thermofisher, Roche).
  • hot-start polymerases and its modifications in various reversibly inactivated states were offered as so-called hot-start Taq polymerase.
  • hot-start polymerases which are converted into an active form, for example by initial heating of the batch at 95 ° C for 1 min to 10 min.
  • the polymerase which has carried out the exponential amplification of the nucleic acid chain to be amplified in the first amplification reaction is inactivated by heating prior to the start of the second amplification.
  • This inactivation can be achieved, for example, by heating the batch to above 80 ° C., better to above 90 ° C., in particular to 95 ° C., for 1 to 10 min.
  • mesophilic polymerases such as Bst polymerase or Bsm polymerase, or Gst polymerase
  • Such inactivation favors conversion of the process from the first amplification reaction to the second amplification reaction.
  • an inactivation of the polymerase of the first amplification reaction can take place simultaneously and an activation of the polymerase for the second amplification reaction.
  • the primer-template complexes are used in particular by the polymerase, which is to carry out the synthesis in the second amplification step.
  • the other polymerase-associated activities such as strand displacement by Bst Polymerase, "turn off”, or "turn on” 5 ' -3 ' -Exonuklease activity of the Taq polymerase.
  • the 5 ' segment of the controller oligonucleotide hybridized to the first primer extension product can be cleaved by the 5 ' -3 nuclease of the polymerase (eg, 5 ' -3 ' nuclease of a Taq polymerase).
  • the polymerase eg, 5 ' -3 ' nuclease of a Taq polymerase.
  • the polymerase can complementarily extend the fourth PCR primer using the third primer extension product under suitable reaction conditions by incorporation of nucleotides and at the same time enzymatically cleave the controller oligonucleotide in its 5 ' segment of the third region hybridized to the third primer extension product.
  • the polymerase-extended strand forms, in particular, an overlap with the controller oligonucleotide by at least one 3 ' -terminal nucleotide during the synthesis, thereby forming a structure recognizable for 5 ' -3 ' -nuclease activity of the Taq polymerase under used reaction conditions, and this structure can be split.
  • 5 ' -3 ' exonuclease activity cleaves the phosphodiester bonds between nucleotides of the third region of the controller oligonucleotide.
  • the 5 ' -3 ' -exonuclease-related cleavage takes place in particular on the DNA strand, and some sugar-phosphate backbone modifications, such as 2 ' -OMe ribonucleotides or PTO modifications or PNA, can slow down or even prevent this cleavage.
  • the controller oligonucleotide in its 5 'segment of the third Area, predominantly by nuclease cleavable nucleotides or their analogs includes (eg DNA).
  • the length of this 5 ' segment of the controller oligonucleotides comprises, for example, 5 to 50, in particular 10 to 30 nucleotides.
  • a polymerase By degrading the controller oligonucleotide bound to the first primer extension product, a polymerase can also extend the second primer without strand displacement properties and thus synthesize the second primer extension product.
  • the presence of components of the second amplification system during the first amplification is considered as follows:
  • the effect of the components of the second amplification system can be reduced by their reversible inactivation prior to the first amplification.
  • individual sequence elements should not occur as nonspecific templates for primer extension during the first amplification. For this reason, primers of the first and second amplification systems should be checked for the existence of self-complementary structures in order to avoid primer-dimer formation.
  • the third PCR primer typically comprises a sequence segment in its 3 ' segment which comprises regions complementary to the controller oligonucleotide. This allows the third PCR Primer to the controller oligonucleotide complementary bind.
  • the third PCR primer must not prevent strand displacement by the controller oligonucleotide during the first amplification.
  • the sequence length or sequence composition of the 3 ' segment of the third PCR primer is adjusted so that it does not bind significantly to the controller oligonucleotide under reaction conditions of the strand displacement step (eg 65 ° C).
  • Such a third PCR primer may further bind with its 3 ' segment to its template and initiate a primer extension reaction.
  • This may be achieved, for example, by the 3 ' segment of the third PCR primer, which may be complementary to the controller oligonucleotide, comprising in its length regions of between 9 and 30 nucleotides, more preferably between 12 and 20 nucleotides , in particular between 12 and 16 nucleotides.
  • This 3 ' segment of the third PCR primer may comprise one or more mismatches to corresponding positions of the controller oligonucleotide such that the binding strength continues to decrease.
  • the sequence segment of the controller oligonucleotide which can substantially complementarily bind the third PCR primer is referred to as the fourth region of the controller oligonucleotide.
  • This fourth region of the controller oligonucleotide may include sequence segments of the second region and / or the third region.
  • the fourth region of the controller oligonucleotide comprises in its length regions which are between 9 and 30 nucleotides, more preferably between 12 and 20 nucleotides, in particular between 12 and 16 nucleotides.
  • the fourth region of the controller oligonucleotide may include one or more mismatches to the 3 ' segment of the third PCR primer.
  • this fourth region comprises nucleotide modifications that prevent the third PCR primer from being extended by a polymerase of the first and / or second amplification system using the controller oligonucleotide as a template. Such modifications have already been described for the combination of the first primer and the controller oligonucleotide.
  • sequence segments 2 '-0-alkyl modifications comprise sequence segments 2 '-0-alkyl modifications, the length of this segment may be 6 to 30 modifications and the complementary nucleotide of the oligonucleotide to the controller 3' -terminal nucleotide of the third PCR primer particular even such modification and further flanked on both sides by at least three nucleotides with such modifications. This ensures that the controller oligonucleotide from the third and fourth primer and the polymerase of the second amplification system is not used as a template.
  • the creation of the first and second primer extension product of a defined length plays a role in the separation of these two products by means of a controller Oligonucleotide. For this reason, it is advantageous to prevent any premature extension of 3 ' ends (each of the first and second primer extension products) using a third or fourth primer of the second amplification system as a template during the first amplification reaction.
  • a reduction or prevention of any undesired excess extension of the 3 ' end of the first primer extension product using the fourth PCR primer as a template can be achieved in various ways: In certain embodiments, this is achieved by the fourth PCR primers, when attached to the 3 ' segment of the first primer extension product, can not undergo perfect match complementary binding with the 3 ' terminal nucleotide of this synthesized first primer extension product. This is intended to prevent or at least reduce excess extension of the 3 ' - terminal nucleotides of the first primer extension product.
  • this is achieved by virtue of the fourth PCR primer, when bound to the 3 ' segment of the first primer extension product, not undergoing perfect match complementary binding with the at least one of the terminal nucleotides of this synthesized first primer extension product can.
  • the resulting at least one mismatch position is in particular in the position of -1 to -5 relative to the terminal nucleotide of the first primer extension product.
  • a reduction or prevention of a possibly unwanted excess extension of the 3 ' end of the second primer extension product using the third PCR primer as a template can be achieved in various ways: In certain embodiments, this is achieved by the third PCR primer, when bound to the 3 ' segment of the second primer extension product, can not undergo perfect match complementary binding with the 3 ' terminal nucleotide of this synthesized first primer extension product. This should an excessive extension of the 3 '- be prevented or at least reduced terminal nucleotides of the second primer extension product.
  • this is accomplished by the third PCR primer, when bound to the 3 ' segment of the second primer extension product, not undergoing perfect match complementary binding with the at least one of the terminal nucleotides of this synthesized second primer extension product can.
  • the resulting at least one mismatch position is in particular in the position -1 to - 5 relative to the terminal nucleotide of the second primer extension product.
  • the presence of components of the first amplification system during the second amplification is considered as follows: On the one hand, the effect of the components of the first amplification system can be reduced by their dilution prior to the second amplification. On the other hand, individual elements should not occur as nonspecific templates for primer extension during the second amplification. For this reason, the primers of the first and second amplification systems should be checked for the presence of self-complementary structures in order to avoid primer-dimer formation.
  • the second amplification is carried out in the presence of the controller oligonucleotide of the first amplification system. For this reason, the design of the controller oligonucleotide, the primer of the second set, and the reaction conditions must be adjusted so that the second amplification reaction is not significantly disturbed by the presence of the controller oligonucleotide.
  • the controller oligonucleotide from both primers and the polymerase from the second amplification system should not be used as a template. This is particularly important for the third oligonucleotide primer, since it comprises a sequence segment which can bind in a complementary manner to the controller oligonucleotide in the fourth region.
  • the structure of the controller oligonucleotides in the fourth region comprises a modification which prevents a potential primer extension. For example, provide such a synthesis blocking effect of various 2 '-0-alkyl modifications.
  • the controller oligonucleotide may form on the third primer extension product during the second amplification and thus on a complementary strand with the third primer extension product.
  • Such a double-stranded fragment may possibly prevent a polymerase from starting to synthesize the complementary strand to the full extent starting from the fourth PCR primer, up to and including the 5 ' segment of the third primer extension product. Therefore, the choice of polymerase and reaction conditions should be such that binding of the controller oligonucleotides does not prevent synthesis of the fourth primer extension product.
  • the length of the segment of the third primer extension product which can make a complementary bond with the controller oligonucleotide is co-determined, for example, by the positioning of the third primer.
  • the third primer is arranged so that the length of the resulting fully complementary portion of an expected third primer extension product to the controller oligonucleotide is in particular in the following ranges: from about 15 nucleotides to about 60 nucleotides, or from about 20 nucleotides to about 40 nucleotides , especially from about 20 nucleotides to about 30 nucleotides.
  • thermostable polymerase is selected in the second amplification reaction which comprises strand displacement activity, for example, a thermostable polymerase such as Vent Exo minus polymerase or pyrophage polymerase can be used.
  • a thermostable polymerase such as Vent Exo minus polymerase or pyrophage polymerase can be used.
  • a polymerase is selected in the second amplification reaction which is capable of cleaving the controller oligonucleotide by 5 ' -3 ' exonuclease activity of the polymerase, with simultaneous primer extension (see above).
  • a controller oligonucleotide is used, which can be cleaved by a 5 ' -3 ' exonuclease, at least in its 5 ' segment.
  • the degradation progressively leads to a shortening of the hybridized to the third primer extension product controller oligonucleotide, which under appropriate reaction conditions (eg, temperature of about 65 ° C to 75 ° C) to destabilize this bond and finally to a separation of the bond to the third primer extension product leads.
  • appropriate reaction conditions eg, temperature of about 65 ° C to 75 ° C
  • the concentration of the controller oligonucleotide and the concentration of the fourth primer of the second primer set are selected such that primer binding and primer extension are faster under the chosen reaction conditions than binding of the controller oligonucleotides to the third primer extension product.
  • concentrations of the controller oligonucleotide in the range of 0.01 pmol / l to 0.3 pmol / l are used.
  • concentrations of the fourth primer ranging from about 0.5 pmol / L to 2 pmol / L are used.
  • polymerases are used in the second amplification reaction, which have high processivity (for example, Phusion Polymerase). The binding of the primer and its extension are generally faster than the binding of the controller oligonucleotides, so that the primer extension reaction is kinetically advantageous.
  • the primer extension reaction in the second amplification reaction is carried out at at least two temperatures.
  • the first, generally lower temperature, eg in the range of 45 ° C to 65 ° C the complementary binding of the fourth primer to its complementary segment occurs in the 3 ' segment of the third primer extension product, as well as an initial extension by the polymerase .
  • a partially extended primer extension product is generated, which has sufficient stability with the third primer extension product, so that after increasing the temperature of the second region, for example to values of about 70 ° C to about 80 ° C, this partially extended fourth Primer extension product can be extended by the polymerase.
  • the controller oligonucleotide can spontaneously dissociate from its binding with the third primer extension product so that the polymerase can continue the synthesis of the fourth primer extension product, up to and including the 5 ' segment of the third primer extension product from the polymerase becomes.
  • polymerases are used in the second amplification reaction, which have a high processivity (eg, Phusion Polymerase). Processes which work with the composition of the reaction mixture in the "homogeneous format" are particularly suitable, for example, if a division of the first reaction batch is not meaningful or technically very complicated, for example in the case of a "micro- or nanotrottle reaction” (also known as called digital PCR).
  • a solid phase comprising oligonucleotides which are suitable for a specific binding of products formed and / or also occur as immobilized primers, so that a solid phase PCR can be carried out, wherein the primer extension products immobilized on the solid phase.
  • the establishment of contact with a solid phase comprising specific oligonucleotides may occur before, during, or only after the first amplification.
  • the reaction mixture can be divided into a plurality of reaction volumes (partitioning) before the start of the first amplification reaction, so that an amplification of a reaction mixture is simultaneously divided into about 100 or 1000 or more equal volume fractions.
  • This division can take place in the form of droplets (for example as an emulsion).
  • the execution of amplification may be carried out in parallel in this plurality of droplets (e.g., as Digital PCR).
  • sequence of the amplification reactions plays an essential role: the sequence-specific amplification (first amplification) using a controller oligonucleotide takes place in particular before a PCR amplification.
  • the PCR-based amplification takes place only as a second step.
  • no DNA fragments generated by a PCR or another amplification method are used as starting nucleic acid chains for the first amplification reaction.
  • the first amplification method (first partial amplification) and the required components of the first amplification system will first be described schematically, then the second amplification method (second partial amplification) and the components of the second amplification system will be described. Subsequently, the combination of the first and the second amplification method, as well as advantageous embodiments are shown.
  • the second amplification method (also referred to below as PCR) takes place after the first amplification.
  • the first amplification occurs as an exponential amplification in which newly synthesized products of both primers (primer extension products) occur as templates for further synthetic steps.
  • the primer sequences are at least partially copied, so that complementary primer binding sites arise, which immediately after their synthesis are present as sequence segments of a double strand.
  • synthesis steps of both strands and double-strand opening steps of the newly synthesized sequence segments take place alternately. Sufficient double-strand separation after synthesis is an important prerequisite for further synthesis. Overall, such a change from synthesis and double-strand separation steps can lead to exponential amplification.
  • the double-stranded opening of the main products of the amplification takes place inter alia by means of an oligonucleotide, which is referred to herein as a controller oligonucleotide.
  • the controller oligonucleotide in particular comprises sequence segments which correspond to the target sequence.
  • the strand separation according to the invention is achieved by using a controller oligonucleotide, each having a predefined sequence, which in particular separates a newly synthesized double strand consisting of two specific primer extension products by means of a sequence-dependent nucleic acid-mediated strand displacement.
  • the resulting single-stranded segments of the primer extension products comprise the target sequence as well as corresponding primer binding sites, which can serve as binding sites for further primer oligonucleotides, so that an exponential amplification of nucleic acid chains to be amplified is achieved.
  • the primer extension reactions and strand displacement reactions take place simultaneously in the approach.
  • the amplification occurs in particular under reaction conditions which do not allow a spontaneous separation of both specific synthesized primer extension products.
  • a specific exponential first amplification of a nucleic acid sequence comprising a target sequence comprises a repetition of synthesis steps and double-strand opening steps (activation steps for primer binding sites) as a mandatory prerequisite for the amplification of the nucleic acid chain.
  • the opening of synthesized duplexes is implemented as a reaction step which is to be sequence-specifically influenced by the controller oligonucleotide. This opening can be complete, even to dissociation of both complementary primer extension products, or even partial.
  • the controller oligonucleotide comprises sequence segments which can interact with the target sequence and further sequence segments which bring about this interaction or facilitate or favor it.
  • double-stranded sections of the synthesized primer extension products are converted into a single-stranded form via sequence-specific strand displacement. This process is sequence-dependent: only when the sequence of the synthesized double strand has a certain degree of complementarity with the corresponding sequence of the controller oligonucleotide does a sufficient double strand opening occur the sequence sections essential for the continuation of the synthesis, such as primer binding sites, are converted into single-stranded form, which corresponds to an "active state".
  • the controller oligonucleotide thus "specifically" activates the newly synthesized primer extension products comprising the target sequence for further synthetic steps.
  • sequence segments which do not comprise a target sequence are not converted into a single-stranded state and remain as a double strand, which corresponds to an "inactive" state.
  • the potential primer binding sites in such a duplex are less favored or completely hindered from interacting with new primers, so that further synthetic steps on such "non-activated" strands generally do not occur.
  • This lack of or decreased activation (i.e., single stranded state) of synthesized nucleic acid strands following a synthesis step results in the successful conclusion that only a reduced amount of primers can successfully participate in a primer extension reaction in the subsequent synthesis step.
  • synthesis steps and activation steps are combined to an amplification process and carried out as many times or repeated until the desired Amount of the specific nucleic acid chain is provided.
  • reaction conditions e.g., temperature
  • the reaction conditions are designed in the course of the first amplification process so that spontaneous separation of complementary primer extension products in the absence of a controller oligonucleotide is unlikely or significantly slowed.
  • the controller oligonucleotide facilitates this double-stranded separation as a result of matching its sequence segments to given sequence segments of the primer extension products. This match is checked after each synthesis cycle by the controller oligonucleotide.
  • the exponential amplification results from successful repeats of syntheses and sequence-specific strand displacements caused by controller oligonucleotide, i. "Activations" (double-stranded openings / double-stranded separations / strand displacement processes resulting in single-stranded form of corresponding primer binding sites) of newly synthesized primer extension products.
  • controller oligonucleotide thus allows a sequence-dependent review of the contents of the primer extension products between the individual synthetic steps during the exponential amplification and inducing a selection of sequences for subsequent synthetic steps. It is possible to distinguish between "active", single-stranded states of newly synthesized specific primers. Extension products can be distinguished as a result of successful interaction with a controller oligonucleotide, and "inactive" double-stranded states of newly synthesized nonspecific primer extension products as a result of deficient, insufficient, decreased and / or slowed interaction with a controller oligonucleotide.
  • the exponential amplification of nucleic acid chains comprising a target sequence is carried out in a sequence-controlled manner (main reaction). This sequence control occurs after each step in the synthesis and includes sequence segments which are between the primers and comprise the target sequence.
  • the successful verification of the result of the synthesis after each synthesis step results in the separation of the two specific primer extension products, which is the prerequisite for further specific synthesis steps.
  • the method thus makes it possible to check the synthesized sequences in real time, ie without reaction interruption, and thus provides means for the development of homogeneous assays in which all components of the assay are already present in the reaction mixture at the beginning of a reaction.
  • PCR second partial amplification
  • the first amplification process is then terminated or the reaction conditions are changed so that the second amplification process (PCR) can be started.
  • the amplification of the target sequences is carried out using nucleic acid chains prepared in the first amplification method and at least one further, PCR-specific component (for example an oligonucleotide primer and / or a PCR polymerase). Since the number of specific nucleic acid chains is sufficiently high already at the beginning of the second amplification, the PCR process requires fewer cycles until the desired total amount of products is reached. This PCR products are generated, which have fewer errors overall.
  • the components of the first amplification system are different from the components of the second amplification system (PCR).
  • the components of the first amplification system are different from the components of the second amplification system, and the PCR components (of the second amplification system) are at least partially in non-active form (eg, hot) under reaction conditions of the first amplification process. Starting polymerases). Switching from the first amplification to the second amplification thus requires an additional activation step of the PCR components (e.g., polymerase).
  • the PCR components e.g., polymerase
  • the polymerase of the first amplification system is inactivated after completion of the first amplification, so that a different polymerase is used for the second amplification (PCR).
  • the components of the first amplification system are also partially utilized by the second amplification system using at least one additional PCR-specific component (e.g., another primer oligonucleotide or polymerase) that is not part of the first amplification system.
  • at least one additional PCR-specific component e.g., another primer oligonucleotide or polymerase
  • the present invention describes some embodiments of both methods, arrays of oligonucleotide primers, which are advantageous for performing both amplification methods.
  • suitable arrangement of individual elements of the first and the second Amplification system on a nucleic acid chain comprising a target sequence homogeneous assays can be assembled with higher overall specificity.
  • oligonucleotide as used herein with reference to primer, controller oligonucleotide, probes, nucleic acid chain to be amplified refers to a molecule having two or more, in particular more than three deoxyribonucleotides and / or ribonucleotides and / or nucleotide modifications and / or or non-nucleotide modifications. Its length includes, for example, ranges between 3 to 300 nucleotide units or its analogs, more preferably between 5 to 200 nucleotide units or its analogs. Its exact size depends on many factors, which in turn depend on the ultimate function or use of the oligonucleotides.
  • primer refers to an oligonucleotide, whether naturally occurring e.g. B. occurs in a purified restriction cleavage or was produced synthetically.
  • a primer is capable of acting as the initiation point of the synthesis when used under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand, ie in the presence of nucleotides and an inducing agent such as DNA polymerase a suitable temperature and a suitable pH.
  • the primer is particularly single-stranded for maximum efficiency in amplification.
  • the primer must be long enough to initiate synthesis of the extension product in the presence of the inducing agent.
  • the exact length of the primer will depend on many factors, including the reaction temperature and primer source, and the application of the method. For example, depending on the complexity of the target sequence, the length of the oligonucleotide primer in diagnostic applications is between 5 to 100 nucleotides, in particular 6 to 40 and particularly preferably 7 to 30 nucleotides. Short primer molecules generally require lower reaction temperatures to perform their primer function to form sufficiently stable complexes with the template, or higher concentrations of other reaction components, such as DNA. Polymerases, so that a sufficient extension of formed primer-template complexes can take place.
  • the primers used herein are selected to be "substantially" complementary to the different strands of each specific sequence to be amplified. That is, the primers must be sufficiently complementary to hybridize with their respective strands and initiate a primer extension reaction. Thus, for example, the primer sequence does not need to reflect the exact sequence of the target sequence.
  • a non-complementary nucleotide fragment may be attached to the 5 'end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • single non-complementary bases or longer non-complementary sequences may be inserted in a primer, provided that the primer sequence has sufficient complementarity with the strand sequence to be amplified to hybridize therewith and thus form a primer template. Complex able to produce the synthesis of the extension product.
  • a primer extension product is generated which is completely complementary to the template strand.
  • the melting temperature of a complementary or partially complementary double strand is generally understood to mean a measured value of a reaction temperature at which approximately half of the strands are in the form of a double strand and the other half is in the form of a single strand.
  • the system association and dissociation of strands is in equilibrium.
  • the determination of the Tm of a nucleic acid to be amplified should take place under the same conditions, like the intended amplification reaction.
  • the melting temperature is understood to mean a value which was measured in the same reaction buffer as the exponential amplification, at concentrations of both complementary components of a double strand of from about 0.1 pmol / l to about 10 pmol / l, in particular in a concentration of about 0.3 pmol / to about 3 pmol / l, in particular about 1 pmol / l.
  • the respective value of the melting temperature is a guideline, which correlates with the stability of a respective double strand.
  • dNTPs deoxyribonucleoside triphosphates
  • dATP deoxyribonucleoside triphosphates
  • dCTP deoxyribonucleoside triphosphates
  • dGTP deoxyribonucleoside triphosphates
  • TTP dUTP, or dUTP / TTP mixture
  • dNTP analogue may be added to the synthesis mixture.
  • these dNTP analogs include a characteristic label (eg, biotin or fluorescent dye) that can be incorporated into the nucleic acid strand.
  • this dNTP analogs include at least one modification of the sugar phosphate moiety of the nucleotide, for example, alpha-phosphorothioate-2 '-Desoxyribonukleosid- triphosphates (or other modifications which impart a nucleic acid strand, a nuclease resistance), 2', 3 'dideoxy-ribonucleoside triphosphates, Azyklo nucleoside triphosphates (or other leading to the termination of a synthetic modifications).
  • alpha-phosphorothioate-2 '-Desoxyribonukleosid- triphosphates or other modifications which impart a nucleic acid strand, a nuclease resistance
  • 2', 3 'dideoxy-ribonucleoside triphosphates or other leading to the termination of a synthetic modifications.
  • these dNTP analogs include at least one modification of nucleobase, eg, iso-cytosines, iso-guanosines (or other modifications of extended-genetic alphabet nucleobases), 2-amino-adenosines, 2-thiouridines, inosines, 7-deazy adenosines, 7-deaza-guanosines, 5-Me-cytosines, 5-propyl-uridines, 5-propyl-cytosines (or other modifications of nucleobases which can be incorporated into natural nucleobases by a polymerase and alter the strand Lead stability).
  • nucleobase eg, iso-cytosines, iso-guanosines (or other modifications of extended-genetic alphabet nucleobases), 2-amino-adenosines, 2-thiouridines, inosines, 7-deazy adenosines, 7-deaza-guanosines, 5-Me-cytosines, 5-propyl-
  • a dNTP analog comprises both a modification of the nucleobase and a modification of the sugar-phosphate moiety.
  • at least one other type of dNTP analogue is added to the synthesis mixture rather than at least one natural dNTP substrate.
  • the nucleic acid synthesis-inducing agent may be an enzyme or a system that functions to cause synthesis of the primer extension products.
  • Suitable enzymes for the first amplification for this purpose include, but are not limited to, DNA polymerases, e.g. Bst polymerase and its modifications, Vent polymerase and others - particularly heat-stable DNA polymerases which allow the incorporation of the nucleotides in the correct manner, thereby forming the primer extension products that are complementary to any nucleic acid strand synthesized.
  • DNA polymerases e.g. Bst polymerase and its modifications, Vent polymerase and others - particularly heat-stable DNA polymerases which allow the incorporation of the nucleotides in the correct manner, thereby forming the primer extension products that are complementary to any nucleic acid strand synthesized.
  • the synthesis is initiated at the 3 'end of each primer and then proceeds in the 5' direction along the template strand until the synthesis is complete or interrupted.
  • template-dependent strand-displaceable DNA polymerases are used in the first amplification. These include, for example, the large fragment of Bst polymerase or its modifications (eg Bst 2.0 DNA polymerase), the Klenow fragment, Vent exo minus polymerase, Deepvent exo minus DNA polymerase, the large fragment of Bsu DNA polymerase, and the large fragment of Bsm DNA polymerase.
  • polymerases which have no 5 ' -3 ' -Exonuklease activity, or have no 5 ' -3 ' -FFEN activity.
  • thermostable matrix-dependent DNA polymerases are used for the second amplification.
  • Taq polymerase or its modifications eg Ampli-Taq
  • polymerases with proofreading function Pfu and its modifications or Vent Polymerase and its modifications.
  • Pfu and its modifications Pfu and its modifications
  • Vent Polymerase and its modifications Pfu and its modifications
  • polymerases which have been fused with another protein for example Phusion Polymerase.
  • Phusion Polymerase Phusion Polymerase.
  • a variety of polymerases are commercially available.
  • Combinations of polymerases may also be used, e.g. OneTaq polymerase (NEB) is a combination of a Taq polymerase and Vent polymerase. Such combinations of polymerases can contribute to higher synthesis accuracy in the second amplification.
  • NEB OneTaq polymerase
  • At least two different polymerases are used, for example, polymerases capable of strand displacement and those having 3 ' -5 ' reproducing activity.
  • polymerases are used with a hot-start function, which can only develop their function when a certain temperature is reached.
  • the first amplification system comprises the components necessary to perform a specific first amplification: the first primer oligonucleotide, the second primer oligonucleotide, the controller oligonucleotide and the first polymerase.
  • nucleotide mixtures and buffer systems may be included to the extent necessary to perform a specific first amplification (e.g., specific buffer systems for the first polymerase).
  • the first amplification system supports the synthesis of the first amplification fragment
  • the first amplification product 1.1 (or of the first amplification product 1.1, also referred to as the nucleic acid sequence of the first amplification to be amplified) during the first amplification (also called the first partial amplification).
  • the second amplification system comprises the components necessary to carry out the specific second amplification: the third primer oligonucleotide, the fourth primer oligonucleotide and the second polymerase.
  • this may include nucleotide mixtures and buffer systems as necessary to perform a specific second amplification (e.g., special buffer systems for the second polymerase).
  • the second amplification system supports synthesis of the second amplification fragment
  • second amplification product 2.1 (or second amplification product 2.1) during the second amplification (also called second partial amplification).
  • First primer oligonucleotide (component of the first amplification system)
  • the first primer oligonucleotide ( Figures 12 to 16) comprises a first primer region and a second region.
  • the first primer region is capable of binding to a substantially complementary sequence within the nucleic acid or its equivalents to be amplified, and a Initiate primer extension reaction.
  • the second region comprises a polynucleotide tail which is capable of binding a controller oligonucleotide and thereby effecting spatial proximity between the controller oligonucleotide and the other portions of the first primer extension product sufficient to induce strand displacement through the controller oligonucleotide.
  • the second region of the first primer oligonucleotide further comprises at least one modification (a nucleotide modification or a non-nucleotide modification) which prevents the polymerase from copying the polynucleotide tail by inhibiting the continuation of the polymerase dependent synthesis.
  • This modification is located, for example, at the junction between the first and second regions of the first primer oligonucleotide.
  • the first primer region of the first primer oligonucleotide is thus replicable by a polymerase so that a complementary sequence to this region can be generated during the synthesis of the second primer extension product (discussed in detail below) from the polymerase.
  • the polynucleotide tail of the second region of the first primer oligonucleotide is not copied by the polymerase. In certain embodiments, this is achieved by the modification in the second region, which stops the polymerase from the polynucleotide tail. In certain embodiments, this is achieved by nucleotide modifications in the second region, where the entire polynucleotide tail consists essentially of such nucleotide modifications and thus is uncopatible to the polymerase.
  • each first primer oligonucleotide is specific for each nucleic acid to be amplified.
  • each first primer oligonucleotide is specific for at least two of the nucleic acids to be amplified, each comprising substantially different sequences.
  • the first primer oligonucleotide is labeled with a characteristic marker, e.g. a fluorescent dye (e.g., TAMRA, fluorescein, Cy3, Cy5) or an affinity tag (e.g., biotin, digoxigenin) or an additional sequence fragment, e.g. for binding a specific oligonucleotide probe for detection or immobilization or barcode labeling.
  • a characteristic marker e.g. a fluorescent dye (e.g., TAMRA, fluorescein, Cy3, Cy5) or an affinity tag (e.g., biotin, digoxigenin) or an additional sequence fragment, e.g. for binding a specific oligonucleotide probe for detection or immobilization or barcode labeling.
  • a first primer oligonucleotide is used in particular in the first amplification as a primer.
  • it may be used as a primer in both the first and second amplifications.
  • Second primer oligonucleotide (component of the first amplification system)
  • primer oligonucleotide capable of binding with its 3 ' segment to a substantially complementary sequence within the nucleic acid or its equivalents to be amplified and initiating a specific second primer extension reaction.
  • primer oligonucleotide is capable of binding to the 3 ' segment of a first specific primer extension product of the first primer oligonucleotide and initiating polymerase-dependent synthesis of a second primer extension product.
  • the length of the second primer oligonucleotide can be between 15 and 100 nucleotides, in particular between 20 and 60 nucleotides, in particular between 30 and 50 nucleotides.
  • every second primer oligonucleotide is specific for each nucleic acid to be amplified.
  • every other primer oligonucleotide is specific for at least two of the nucleic acids to be amplified, each comprising different sequences.
  • the second primer oligonucleotide is labeled with a characteristic marker, e.g. a fluorescent dye (e.g., TAMRA, fluorescein, Cy3, Cy5) or an affinity tag (e.g., biotin, digoxigenin) or an additional sequence fragment, e.g. for binding a specific oligonucleotide probe for detection or immobilization or barcode labeling.
  • a characteristic marker e.g. a fluorescent dye (e.g., TAMRA, fluorescein, Cy3, Cy5) or an affinity tag (e.g., biotin, digoxigenin) or an additional sequence fragment, e.g. for binding a specific oligonucleotide probe for detection or immobilization or barcode labeling.
  • a second primer oligonucleotide is used in particular in the first amplification as a primer. In certain embodiments, it may be used as a primer in both the first and second amplifications.
  • a primer extension product results from enzymatic (polymerase-dependent) extension of a primer oligonucleotide as a result of template-dependent synthesis catalyzed by a polymerase.
  • a primer extension product comprises the sequence of the primer oligonucleotide in its 5 ' segment and the sequence of the extension product (also called extension product) which has been synthesized by a polymerase in a template-dependent form.
  • the extension product synthesized by the polymerase is complementary to the template strand on which it was synthesized.
  • a specific primer extension product (eg P1 .1-Ext and P2.1-Tex) of the first amplification (main product) comprises sequences of the nucleic acid chain to be amplified. It is the result of a specific synthesis or a correct execution of an intended primer extension reaction in which the nucleic acid chain to be specifically amplified serves as a template.
  • the sequence of the synthesized primer extension products coincides completely with the expected sequence of a nucleic acid to be amplified. In another embodiment, deviations in the sequence obtained may be tolerated by the theoretically expected sequence.
  • the degree of agreement of the sequence obtained is due to a Amplification with the sequence of the theoretically expected to be amplified nucleic acid, for example, between 90% and 100%, in particular, the match is over 95%, ideally, the agreement is greater than 98% (measured by the proportion of synthesized bases).
  • the length of the extension product of a specific primer extension product can be between 10 and 300 nucleotides, in particular between 10 and 180 nucleotides, in particular between 20 and 120 nucleotides, in particular between 30 and 80 nucleotides.
  • products of a specific template-dependent primer extension reaction of the third and fourth primers represent the major products or intermediates: the partial third primer extension product (P3.1-Ext Part 1, Figs. 15-18). or the complete primer extension product (P3.1-Ext, FIGS. 15-18), the partial fourth primer extension product (P4.1-Ext Part 1, FIGS. 15-18), or the complete primer Extension product (P4.1 -Ext, Figs. 15-18).
  • These products in certain embodiments, comprise a target sequence or its equivalents.
  • the second primer extension product comprises a target sequence and the first primer extension product comprises equivalents of the target sequence, namely the complementary strand.
  • the fourth primer extension product comprises a target sequence and the third primer extension product comprises equivalents of the target sequence, the complementary strand. In certain embodiments, the fourth primer extension product comprises portions of a target sequence and the third primer extension product comprises equivalents of these portions of the target sequence, namely the complementary strand.
  • the first primer extension product (P1 .1-Ext) and the second primer extension product (P2.1-Ext) together represent the first amplification fragment 1 .1 (also referred to as amplification product 1.1) of the first amplification (Fig . 1 ).
  • the third primer extension product (P3.1 -Ext) and the fourth primer extension product (P4.1-Ext) together constitute the second amplification fragment 2.1 (also referred to as the second amplification product 2.1) of the second amplification (Fig . 1 ).
  • the third primer extension product (P3.1 Ext) may also be referred to as a complete third primer extension product. This product is made using the fourth primer extension product (P4.1-Ext) as template.
  • the fourth primer extension product (P4.1-Ext) may also be referred to as a complete fourth primer extension product. This product is formed using the third primer extension product (P3.1-Ext) as template ( Figures 15-18).
  • a non-specific primer extension product includes sequences that have resulted as a result of a nonspecific or improper primer extension reaction. These include, for example, primer extension products that have arisen as a result of a false initiation event (false priming) or as a result of other side reactions, including polymerase-dependent sequence changes such as base substitution, deletion, etc.
  • the level of sequence aberrations of nonspecific primer extension products generally exceeds the ability of controller oligonucleotides to successfully displace such double-stranded by-products from their templates so that amplification of such by-products is slower or completely absent.
  • the extent of acceptance or the tolerance limit for deviations depend, for example, on the reaction temperature and the manner of sequencing deviation.
  • examples of nonspecific primer extension products are primer dimers or sequence variants which do not correspond to the nucleic acid to be amplified, e.g. Sequences which do not comprise a target sequence.
  • the assessment of a sufficient specificity of the amplification is often related to the task. In many amplification methods, some degree of unspecificity of the amplification reaction can be tolerated as long as the desired result can be achieved. In a particular embodiment, the proportion of nucleic acid chains to be amplified in the overall result of the reaction is more than 1%, in particular more than 10%, in particular more than 30%, based on the total amount of newly synthesized strands.
  • the nucleic acid to be amplified (expected result of the first amplification)
  • the nucleic acid to be amplified represents a nucleic acid chain which is to be amplified sequence-specifically or at least predominantly sequence-specifically by means of the first amplification using primers and controller oligonucleotide by the polymerase.
  • the nucleic acid to be amplified comprises both strands of the first amplification fragment 1 .1 (also referred to as first amplification product 1.1) (FIG. 1). It can be used as starting nucleic acid chain 2.1, wherein at least one of its strands can be used to initiate the second amplification.
  • the length of the nucleic acid to be amplified may be between 20 and 300 nucleotides, in particular between 30 and 200 nucleotides, in particular between 40 and 150 nucleotides, in particular between 50 and 100 nucleotides.
  • the nucleic acid sequence to be amplified may comprise one or more target sequences or their equivalents.
  • a nucleic acid to be amplified may comprise the sequences substantially complementary to a target sequence, which are propagated with similar efficiency as a target sequence in an amplification reaction and the target sequence or its Sections include.
  • the nucleic acid to be amplified may include additional sequence segments, for example, primer sequences, sequences with primer binding sites and / or sequence segments for binding of detection probes, and / or sequence segments for sequence encoding of strands by barcode sequences and / or sequence segments for binding to a solid phase.
  • the primer sequences or their sequence portions, as well as primer binding sites or their sequence portions may for example belong to sequence sections of a target sequence.
  • the nucleic acid to be amplified corresponds to the target sequence.
  • the target sequence forms part of the sequence of the nucleic acid sequence of the first amplification to be amplified.
  • a target sequence may be flanked by 3 ' side and / or 5 ' side of further sequences.
  • These further sequences may include, for example, binding sites for primers or their moieties, and / or comprising primer sequences or their moieties, and / or binding sites for detection probes, and / or adapter sequences for complementary binding to a solid phase (eg, Im Frame of microarrays, or bead-based analyzes) and / or include barcoding sequences for a digital signature of sequences.
  • a nucleic acid chain In order for the amplification to start, a nucleic acid chain must be added to the reaction mixture at the beginning of the reaction, which occurs as an initial template for the synthesis of the nucleic acid chain to be amplified. This nucleic acid chain is called the start nucleic acid chain. This start nucleic acid chain predetermines the arrangement of individual sequence elements which are important for the formation / synthesis / exponential amplification of a nucleic acid chain to be amplified.
  • the initial template (starting nucleic acid chain), which is supplied to an amplification reaction at the beginning, or which is added to the reaction mixture, corresponds to the sequence composition of the nucleic acid chain to be amplified.
  • the respective primers bind to the corresponding binding sites in the starting nucleic acid chain and initiate the synthesis of specific primer extension products.
  • specific primer extension products accumulate exponentially in the course of amplification and increasingly assume the role of templates for the synthesis of complementary primer extension products in exponential amplification.
  • the nucleic acid chain to be amplified is thus formed.
  • the major product of the reaction (the nucleic acid to be amplified) may be predominantly single-stranded or predominantly a complementary duplex. This can be done, for example, by the relative Concentrations of both primers and corresponding reaction conditions can be determined.
  • nucleic acid to be amplified include nucleic acids having substantially identical information content.
  • complementary strands of a nucleic acid to be amplified have identical information content and can be said to be equivalent.
  • a target sequence is a segment of a nucleic acid chain to be amplified which can serve as a characteristic sequence of the nucleic acid to be amplified. This target sequence can serve as a marker for the presence or absence of another nucleic acid.
  • This other nucleic acid thus serves as the source of the target sequence and can be for example a genomic DNA or RNA or parts of the genomic DNA or RNA (eg mRNA), or equivalents of the genomic DNA or RNA of an organism (eg cDNA, modified RNA such as rRNA, tRNA, microRNA, etc.), or defined changes in the genomic DNA or RNA of an organism, for example mutations (eg deletions, insertions, substitutions, additions, sequence amplification, eg repeat propagation in the context of microsatellite instability), splice variants, rearrangement variants (eg, T cell receptor variants), etc.
  • mutations eg deletions, insertions, substitutions, additions, sequence amplification, eg repeat propagation in the context of microsatellite instability
  • splice variants eg, rearrangement variants (eg, T cell receptor variants), etc.
  • the individual target sequences may represent a phenotypic trait, such as antibiotic resistance or prognostic information, and thus be of interest for diagnostic assays / assays.
  • a source / origin of a target sequence such a nucleic acid may comprise, for example, the target sequence as a sequence element of its strand.
  • a target sequence can thus serve as a characteristic marker for a particular sequence content of another nucleic acid.
  • the target sequence can be single-stranded or double-stranded. It may be substantially identical to the nucleic acid of the first amplification to be amplified or may be only part of the nucleic acid to be amplified.
  • Equivalents of the target sequence include nucleic acids with substantially identical information content.
  • complementary strands of a target sequence have identical informational content and can be said to be equivalent;
  • RNA and DNA variants of a sequence are also examples of equivalent informational content.
  • such a target sequence can be isolated from its original sequence environment and prepared for the amplification reaction.
  • a nucleic acid to be amplified comprises a target sequence.
  • the target sequence corresponds to the nucleic acid to be amplified.
  • a startup Nucleic acid chain 1 .1 a target sequence.
  • the target sequence corresponds to a starting nucleic acid chain 1 .1
  • the target sequence may be fully or partially included during the first amplification as part of the first amplification fragment 1 .1.
  • the second primer extension product comprises the target sequence or its portions and the second primer extension product thus comprises the strand complementary to this target sequence.
  • This strand has an equivalent information content.
  • the target sequence may be wholly or partially included during the second amplification as part of the second amplification fragment 2.1.
  • the fourth primer extension product comprises the target sequence or its portions, and the third primer extension product thus comprises the strand complementary to this target sequence.
  • This strand has an equivalent information content.
  • a nucleic acid chain In order for the first amplification to start, a nucleic acid chain must be added to the reaction mixture at the beginning of the reaction which occurs as an initial template for the synthesis of the nucleic acid chain to be amplified (FIG. 1).
  • This nucleic acid chain is referred to as the starting nucleic acid chain of the first amplification (starting nucleic acid chain 1.1).
  • This start nucleic acid chain predetermines the arrangement of individual sequence elements which are important for the formation / synthesis / exponential amplification of a nucleic acid chain to be amplified.
  • Such a starting nucleic acid chain can be present in single-stranded or double-stranded form at the beginning of the reaction.
  • the complementary strands of the starting nucleic acid chain are separated, regardless of whether the nucleic acid was originally double- or single-stranded, the strands can serve as a template for the synthesis of specific complementary primer extension products.
  • the starting nucleic acid chain 1 .1 comprises, in certain embodiments, a target sequence.
  • a start nucleic acid chain furthermore comprises at least one predominantly single-stranded sequence segment, to which at least one of the primers of the amplification system can bind predominantly complementarily with its 3 ' segment, so that the polymerase used contains such a primer, when hybridized to the starting nucleic acid chain, may extend template-specific incorporation of dNTPs.
  • the start-up nucleic acid chain may also comprise segments which are not the target sequence but which can bind primers.
  • a nucleic acid chain In order for the second amplification to start, at the beginning of the second amplification reaction, a nucleic acid chain must be present in the reaction mixture or be added thereto, which occurs as an initial template for the synthesis of the second amplification fragment (FIG. 1).
  • This nucleic acid chain is referred to as the starting nucleic acid chain of the second amplification (starting nucleic acid chain 2.1).
  • This start nucleic acid chain 2.1 specifies the arrangement of individual sequence elements which are important for the formation / synthesis / exponential amplification of a nucleic acid chain to be amplified.
  • the first amplification fragment 1.1 generated during the first amplification serves as starting nucleic acid chain 2.1 for the second amplification.
  • This start nucleic acid chain 2.1 thus comprises or consists of the first primer extension product produced in the first amplification.
  • the starting nucleic acid chain 2.1 comprises in certain embodiments a target sequence.
  • PCR amplification also called second amplification
  • PCR is a polymerase chain reaction which generally serves to amplify DNA fragments. Many variants of a PCR reaction are known in the art. Among other things, allel-specific PCR, genotyping PCR, assymetric PCR, solid-phase PCR, digital PCR (partitioning / microdroplet PCR), multiplex PCR, real-time PCR using probes or intercalating dyes (eg Molecular beacons or 5 ' -3 ' exonuclease or two oligonucleotide probes with FRET pairs), clamp PCR using blocking probes, quantitative PCR, nested PCR, etc.
  • probes or intercalating dyes eg Molecular beacons or 5 ' -3 ' exonuclease or two oligonucleotide probes with FRET pairs
  • clamp PCR using blocking probes
  • quantitative PCR quantitative PCR
  • nested PCR etc.
  • a low temperature temperature range for binding of primers to their predominantly specific sequence segments and extension to form a complementary strand at least one temperature range involving predominantly sequence-unselective separation of Strands are made so that freshly synthesized primer extension products separate from their templates.
  • PCR can be performed in liquid phase using PCR tubes / microtiter plates or on a solid phase or in microfluidic systems or in situ.
  • PCR primer pair (the third and the fourth primer oligonucleotide)
  • a PCR primer pair typically includes two oligonucleotides capable of supporting amplification of a nucleic acid fragment by a PCR method.
  • a PCR primer pair comprises a third and a fourth primer oligonucleotide.
  • Such primers are known in the art.
  • PCR primers are generally referred to herein as P3 and P4 (as opposed to a controller Abhangige Amplification P1 and P2).
  • PCR primers are known to a person skilled in the art. Typically, they are oligonucleotides of about 15 to about 60 nucleotides in length, which comprise at least in their 3 ' segment sequences capable of binding to complementary segments of a target sequence and using this target sequence as a template to initiate template-dependent synthesis by means of a polymerase.
  • PCR primers may be used in solution or also coupled to a solid phase (e.g., beads, micrititer plates). These variants are also well known to a person skilled in the art.
  • the controller oligonucleotide ( Figure 1, C1. 1) is a single-stranded nucleic acid chain which includes a predefined substantially complementary sequence in a portion of the first primer extension product which is specifically generated in the amplification of the nucleic acid to be amplified (first amplification ). This allows the controller oligonucleotide to bind substantially complementary to the first primer oligonucleotide and at least to the 5 ' segment of the specific extension product of the first primer oligonucleotide.
  • the controller oligonucleotide in its inner sequence segment comprises nucleotide modifications that prevent the polymerase from synthesizing a complementary strand using the controller oligonucleotide as a template when the first and / or third primer oligonucleotide is attached to the controller Oligonucleotide is bound complementarily.
  • the controller oligonucleotide consists entirely of modified nucleotides that block its template function.
  • it is advantageous if the controller oligonucleotide consists only in part of modified nucleotides that block its template function.
  • the controller oligonucleotide is further capable of completely or partially displacing the second specific primer extension product from binding with the first specific primer extension product under strand reaction under the chosen reaction conditions.
  • the controller oligonucleotide with its complementary regions attaches itself to the first specific primer extension product.
  • this leads to recovery of a single-stranded state of the 3 ' segment of the second specific primer extension product suitable for binding the first primer oligonucleotide so that a new primer extension reaction can take place.
  • the controller oligonucleotide may be separated from binding with the first primer extension product by strand displacement, for example, by polymerase and / or by the second primer oligonucleotide.
  • a detection system comprises at least one oligonucleotide probe and at least one fluorescent reporter (a fluorophore).
  • the detection system should be capable of carrying out the synthesis of the first and / or the second and / or the third and / or the fourth primer extension product (P1 1-Ext, P2.1-Ext, P3.1-Ext, P4. 1-text). This is done by using oligonucleotide probes, which are able to bind to the respective primer extension product and thereby generate a specific signal, or bring about a change in the signal. This change may be an increase or decrease in fluorescence intensity.
  • a detection system may further comprise additional components. Such components include in particular fluorescence quencher and / or donor fluorophore.
  • the detection system may also include the controller oligonucleotide.
  • the array of fluorescent reporter, fluorescence quencher, donor fluorophore on the oligonucleotide probe or on the pair comprising oligonucleotide probe and controller oligonucleotide makes it possible to detect the binding events to the first primer extension product.
  • a fluorophore is a chemical compound or molecule which, when excited by electromagnetic radiation, is capable of emitting electromagnetic radiation (light) (emission). This radiation emitted by the fluorophore (emission) can be detected as a fluorescence signal by suitable technical means.
  • a reporter can be covalently bound to an oligonucleotide.
  • fluorophores are known which can be coupled to oligonucleotides (e.g., FAM, TAMRA, HEX, ROX, Cy dyes, Alexa dyes).
  • a quencher is a chemical compound / molecule capable of reducing the emission of a fluorophore by direct contact (contact quenching) or by energy transfer (eg, FRET).
  • a quencher must be placed in close proximity to the fluorophore for significant signal attenuation.
  • Advantageous for a significant signal reduction of a fluorescence reporter by a quencher are distances between the reporter and quencher of less than 25 nucleotides, in particular of less than 15 nucleotides, in particular of less than 5 nucleotides.
  • the distance between these components must be increased accordingly, it is advantageous if the distance between the reporter and quencher of more than 15 nucleotides, in particular more than 20 nucleotides, in particular more than 40 nucleotides is increased.
  • the distance caused by the nucleotide sequence must be due to an extended nucleotide sequence. For example, if hairpin structures form, spatial distance may no longer exist.
  • fluorophore-quencher pairs are preferred which have more than 25% spectral overlap. (e.g., FAM / TAMRA).
  • Donor fluorophores are chemical compounds / molecules that are capable of absorbing electromagnetic radiation and transferring it to another fluorophore (acceptor) through energy transfer (eg, as FRET) to excite this fluorophore, and as a result, itself Light emission generated. Upon emission, a fluorescence signal is generated. As a rule, the donor and the acceptor form a fluorescence resonance energy transfer pair (a FRET pair). In general, a donor must be placed in close proximity to the acceptor (fluorophore) for this signal generation to occur to any significant extent.
  • FRET fluorescence resonance energy transfer pair
  • distances between the reporter and donor are distances between the reporter and donor of less than 25 nucleotides, in particular less than 15 nucleotides, in particular less than 5 nucleotides.
  • the removal of the FRET effect from donor to acceptor is usually achieved by increasing the distance between both partners of a FRET pair. It is advantageous if the distance between the reporter and donor is increased to more than 15 nucleotides, in particular to more than 20 nucleotides, in particular to more than 40 nucleotides.
  • the spatial distance between the individual components is brought about, in particular, by connecting two components via a nucleotide sequence which can hybridize with a specific part of the target sequence.
  • a suitable agent leads to a complete or partial separation of a first double strand (consisting for example of A1 and B1 strand) and for the simultaneous / parallel formation of a new second double strand, wherein at least one of the strands ( A1 or B1) is involved in the formation of this new second strand.
  • the formation of a new second duplex may be accomplished using an already existing complementary strand which is generally in single-stranded form at the beginning of the reaction.
  • the means of strand displacement for example, a preformed single-stranded strand C1, which has a complementary sequence to the strand A1, acts on the first already formed double strand (A1 and B1) and enters into a complementary bond with the strand A1, whereby the strand B1 aus
  • the displacement of B1 is completed, the result of the C1 action is a new duplex (A1: C1) and a single strand B1.
  • the displacement of B1 is incomplete, it depends
  • a complex of partially double-stranded A1: B1 and A1: C1 may be present as an intermediate.
  • the formation of a new second duplex may occur under concurrent enzymatic synthesis of the complementary strand, with one strand of the first preformed duplex appearing as a template for synthesis by the polymerase.
  • the means of strand displacement acts on the first already preformed duplex (A1 and B1) and synthesizes a complementary to strand A1 complementary strand D 1, wherein at the same time the strand B1 from the bond with the strand A1 is displaced.
  • nucleic acid mediated strand displacement is meant a sum / series of intermediate steps which can be in equilibrium with one another and, as a result, the temporary or permanent opening of a first preformed duplex (consisting of complementary strands A1 and B1) and formation of a new one second duplex (consisting of complementary strands A1 and C1), wherein A1 and C1 are complementary to each other.
  • an essential structural prerequisite for the initiation of strand displacement is the creation of a spatial proximity between a duplex end (preformed first duplex of A1 and B1) and a single-stranded strand (C1) which initiates strand displacement (where A1 and C1 can form a complementary strand).
  • Such spatial proximity can be brought about in particular by means of a single-stranded overhang (in the literature examples are known with short overhangs, referred to in English as "Toehold", see above), which binds the single-stranded strand (C1) temporarily or permanently complementary, and thus brings complementary Segqmente the strand C1 and A1 sufficiently close, so that a successful strand displacement of the strand B1 can be initiated.
  • the efficiency of initiation of nucleic acid-mediated strand displacement is generally greater the closer the complementary segments of strand A1 and C1 are positioned to each other.
  • nucleic acid-mediated strand displacement in internal segments Another essential structural prerequisite for efficiently continuing nucleic acid-mediated strand displacement in internal segments is high complementarity between strands (e.g., between A1 and C1) which must form a new duplex.
  • strands e.g., between A1 and C1
  • single nucleotide mutations in C1 can lead to the disruption of strand displacement (described, for example, for branch migration).
  • the present invention makes use of the ability of complementary nucleic acids to sequence-dependent nucleic acid-mediated strand displacement.
  • Reaction conditions include, but are not limited to, buffer conditions, temperature conditions, duration of reaction, and concentrations of respective reaction components.
  • the amount of specific produced nucleic acids to be amplified accumulates in an exponential manner.
  • the reaction comprising the synthesis of the extension products can be carried out for as long as necessary to produce the desired amount of the specific nucleic acid sequence.
  • the inventive method is carried out in particular continuously.
  • the amplification reaction proceeds at the same reaction temperature, wherein the temperature is in particular between 50 ° C and 70 ° C.
  • the reaction temperature can also be controlled variably, so that individual steps of the amplification run at respectively different temperatures.
  • reagents required for the exponential amplification are in particular already present at the beginning of a reaction in the same batch.
  • reagents may also be added at later stages of the process.
  • no helicases or recombinases are used in the reaction mixture to separate the newly synthesized duplexes of the nucleic acid to be amplified.
  • the reaction mixture does not include biochemical energy-donating compounds such as ATP.
  • the amount of nucleic acid to be amplified at the beginning of the reaction can be between a few copies and several billion copies in one run.
  • the amount of nucleic acid chain to be amplified may be unknown.
  • the reaction may also contain other nucleic acids which are not to be amplified. These nucleic acids may be derived from natural DNA or RNA or their equivalents. In certain embodiments, control sequences are in the same approach, which should also be amplified in parallel to the nucleic acid to be amplified.
  • a molar excess of about 10 3 : 1 to about 10 15 : 1 (primer: template ratio) of the primers used and the controller oligonucleotide is added to the reaction mixture, which comprises template strands for the synthesis of the nucleic acid sequence to be amplified.
  • the amount of target nucleic acids may not be known if the method of the invention is used in diagnostic applications, so that the relative amount of the primer and the controller oligonucleotide relative to the complementary strand can not be determined with certainty.
  • the amount of primer added will generally be present in molar excess relative to the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.
  • the concentrations of primer 1, primer 2 and controller oligonucleotides used are, for example, in the range between 0.01 pmol / l and 100 pmol / l, in particular between 0.1 pmol / l and 100 pmol / l, in particular between 0, 1 pmol / l and 50 pmol / l, in particular between 0.1 pmol / l and 20 pmol / l.
  • the high concentration of components can increase the rate of amplification.
  • the respective concentrations of individual components can be varied independently of one another in order to achieve the desired reaction result.
  • the concentration of polymerase ranges between 0.001 pmol / l and 50 pmol / l, in particular between 0.01 pmol / l and 20 pmol / l, in particular between 0.1 pmol / l and 10 pmol / l.
  • the concentration of individual dNTP substrates is in the range between 10 pmol / l and 10 mmol / l, in particular between 50 pmol / l and 2 mmol / l, in particular between 100 pmol / l and 1 mmol / l.
  • the concentration of dNTP can affect the concentration of divalent metal cations. If necessary, this will be adjusted accordingly.
  • divalent metal cations Mg2 +, for example, are used.
  • As a corresponding anion for example, CI, acetate, sulfate, glutamate, etc. can be used.
  • the concentration of divalent metal cations is adapted, for example, to the optimum range for the particular polymerase and comprises ranges between 0.1 mmol / l and 50 mmol / l, more preferably between 0.5 mmol / l and 20 mmol / l, more preferably between 1 mmol / l and 15 mmol / l.
  • the enzymatic synthesis is generally carried out in a buffered aqueous solution.
  • buffer solutions dissolved conventional buffer substances, such as Tris-HCl, Tris-acetate, potassium glutamate, HEPES buffer, sodium glutamate in conventional concentrations can be used.
  • the pH of these solutions is usually between 7 and 9.5, in particular about 8 to 8.5.
  • the buffer conditions can be adapted, for example, according to the recommendation of the manufacturer of the polymerase used.
  • Tm depressants e.g., DMSO, betaines, TPAC
  • Tm depressors e.g., DMSO, betaines, TPAC
  • Tween 20 or T riton 100 can also be added in conventional amounts to the buffer.
  • EDTA or EGTA can be added to complex heavy metals in conventional amounts.
  • Polymerase stabilizing substances such as trehalose or PEG 6000 may also be added to the reaction mixture.
  • the reaction mixture contains no inhibitors of the strand displacement reaction and no inhibitors of polymerase-dependent primer extension.
  • the reaction mixture contains DNA-binding dyes, especially intercalating dyes, such as e.g. EvaGreen or SybrGreen.
  • intercalating dyes such as e.g. EvaGreen or SybrGreen.
  • Such dyes may possibly enable the detection of the formation of new nucleic acid chains.
  • the reaction mixture can furthermore contain proteins or other substances which, for example, originate from an original material and which do not influence the amplification.
  • the temperature has a significant influence on the stability of the double strands.
  • no temperature conditions are used during the amplification reaction, which essentially result in the separation of double strands of the nucleic acid to be amplified in the absence of controller oligonucleotide. This is to ensure that the double-stranded separation of nucleic acid chains to be amplified is dependent on the presence of the controller oligonucleotide throughout the course of the amplification.
  • the reaction temperature may be around the melting temperature (i.e., Tm plus / minus 3 ° to 5 ° C) of the nucleic acid to be amplified.
  • Tm melting temperature
  • the reaction temperature may be around the melting temperature (i.e., Tm plus / minus 3 ° to 5 ° C) of the nucleic acid to be amplified.
  • sequence differences between the controller oligonucleotide and the synthesized primer extension product can generally be well tolerated in a strand displacement reaction.
  • controller oligonucleotide On the sequence specificity of the nucleic acid to be amplified is higher than at temperature conditions around the melting temperature (Tm) of the nucleic acid chain to be amplified.
  • primer duplexes may spontaneously decay at the extension temperature under the conditions mentioned; without sequence-dependent strand displacement by controller oligonucleotide.
  • the reaction temperature with a reduced sequence-specific amplification in ranges between about (Tm minus 3 ° C) and about (Tm minus 10 ° C), in particular between about (Tm minus 5 ° C) and about (Tm minus 10 ° C). At such temperature, sequence differences between controller oligonucleotide and the synthesized primer extension product are less tolerated in a strand displacement reaction.
  • a high sequence specificity of the amplification of the method is achieved, in particular, when the newly synthesized strands of the nucleic acid to be amplified can not spontaneously dissociate into single strands under reaction conditions.
  • the sequence-specific strand displacement by controller oligonucleotide plays a crucial role in sequence-specific strand separation and is significantly responsible for the sequence specificity of the amplification reaction. This can generally be achieved if the reaction temperature is well below the melting temperature of both strands of the nucleic acid to be amplified and no further components are used for strand separation, for example no helicases or recombinases.
  • the reaction temperature in a sequence-specific amplification in ranges between about (Tm minus 10 ° C) and about (Tm minus 50 ° C), in particular between about (Tm minus 15 ° C) and about (Tm minus 40 ° C), in particular between about (Tm minus 15 ° C) and about (Tm minus 30 ° C).
  • the maximum reaction temperature in the course of the entire amplification reaction is not increased above the melting temperature of the nucleic acid chain to be amplified.
  • the reaction temperature may be raised at least once above the melting temperature of the nucleic acid chains to be amplified.
  • the increase in temperature can be carried out, for example, at the beginning of the amplification reaction and lead to the denaturation of double strands of a genomic DNA. It should be noted that during such a step, the dependence of the double strand separation on the effect of the controller oligonucleotide is canceled or at least significantly reduced.
  • the reaction temperatures of the individual steps of the amplification reaction can be in the range of about 15 ° C to about 85 ° C, more preferably in the range of about 15 ° C to about 75 ° C, in particular in the range of about 25 ° C to about 70 ° C.
  • a reaction temperature of the amplification reaction of 65 ° C was used, wherein the Tm of the nucleic acids to be amplified between about 75 ° C and about 80 ° C.
  • the duplex of the nucleic acid to be amplified was stable under reaction conditions and the amplification reaction sequence-specific.
  • the reaction temperature can be optimally adjusted for each individual reaction step, so that such a temperature is brought about for each reaction step.
  • the amplification reaction thus comprises a repetitive change of temperatures, which are repeated cyclically.
  • reaction conditions are standardized for a plurality of reaction steps, so that the number of temperature steps is less than the number of reaction steps.
  • at least one of the steps of the amplification takes place at a reaction temperature which differs from the reaction temperature of other steps of the amplification. The reaction is thus not isothermal, but the reaction temperature is changed cyclically.
  • the lower temperature range includes, for example, temperatures between 25 ° C and 60 ° C, in particular between 35 ° C and 60 ° C, in particular between 50 ° C and 60 ° C and the upper temperature range includes, for example, temperatures between 60 ° C and 75 ° C, especially between 60 ° C and 70 ° C.
  • the lower temperature range includes, for example, temperatures between 15 ° C and 50 ° C, in particular between 25 ° C and 50 ° C, in particular between 30 ° C and 50 ° C and the upper temperature range includes, for example, temperatures between 50 ° C and 75 ° C, in particular between 50 ° C and 65 ° C.
  • the lower temperature range includes, for example, temperatures between 15 ° C and 40 ° C, in particular between 25 ° C and 40 ° C, in particular between 30 ° C and 40 ° C and the upper temperature range includes, for example, temperatures between 40 ° C and 75 ° C, especially between 40 ° C and 65 ° C.
  • the temperature can be kept constant in the respective range or can be changed as a temperature gradient (decreasing or rising).
  • Any induced temperature can be maintained for a period of time, resulting in an incubation step.
  • the reaction mixture may thus be incubated for a period of time during amplification at a selected temperature. This time may be different for the particular incubation step and may be responsive to the progress of the particular reaction at a given temperature (e.g., primer extension or strand displacement, etc.).
  • the time of an incubation step may comprise the following ranges: between 0.1 sec and 10,000 sec, in particular between 0.1 sec and 1000 sec, in particular between 1 sec and 300 sec, in particular between 1 sec and 100 sec.
  • a temperature change By means of such a temperature change, individual reaction steps can be carried out, in particular at a selected temperature. As a result, yields of a particular reaction step can be improved.
  • temperature changes or temperature changes between individual temperature ranges may be required several times.
  • a synthesis cycle may thus comprise at least one temperature change. Such a temperature change can be performed routinely, for example, in a PCR device / thermocycler as a time program.
  • an amplification method is preferred in which at least one of the steps comprising strand displacement and at least one of the steps comprising primer extension reactions take place simultaneously and in parallel and under the same reaction conditions.
  • a primer extension reaction of at least one primer oligonucleotide e.g., of the first primer oligonucleotide
  • the strand displacement is carried out with the aid of controller oligonucleotide and the one further primer extension reaction (for example, by the second primer oligonucleotide), in particular in the reaction step in the upper temperature range.
  • an amplification method is preferred in which at least one of the steps comprising strand displacement by the controller oligonucleotide and at least one of the steps comprising primer extension reaction is included performed different temperatures.
  • primer extension reactions of at least one primer oligonucleotide eg, of the first primer oligonucleotide and / or of the second primer oligonucleotide
  • the strand displacement takes place with the assistance of controller oligonucleotide, in particular in the reaction step in the upper temperature range.
  • the steps of an amplification reaction proceed under identical reaction conditions.
  • the amplification process may be carried out under isothermal conditions, i. that no temperature changes are required to carry out the process.
  • the entire amplification reaction is carried out under constant temperature, i. the reaction is isothermal.
  • the time of such a reaction comprises, for example, the following ranges: between 100 sec and 30,000 sec, in particular between 100 sec and 10,000 sec, in particular between 100 sec and 1000 sec.
  • a synthesis cycle The sum of all process steps, which leads to a doubling of the amount of a nucleic acid chain to be amplified, can be referred to as a synthesis cycle.
  • Such a cycle can be correspondingly isothermal or else characterized by changes in the temperature in its course. The temperature changes can be repeated from cycle to cycle and made identical.
  • amplification methods in which the maximum achievable temperature essentially only permits a strand separation with the aid of controller oligonucleotide if more than 5 nucleotides of the third region of the controller oligonucleotide can form a complementary bond with the first primer extension product especially if more than 10, especially if more than 20 nucleotides of the controller oligonucleotide bind with the first primer extension product.
  • the desired level of specificity can be determined.
  • a process step can be carried out at its constant temperature repetition over the entire duration of the process or at different temperatures. Individual process steps can each be carried out in succession by adding individual components. In a particular embodiment, all reaction components necessary for the execution of an amplification are present at the beginning of an amplification in a reaction mixture.
  • the start of an amplification reaction can be carried out by adding a component, e.g. by adding a nucleic acid chain comprising a target sequence (e.g., a starting nucleic acid chain), or a polymerase or divalent metal ions, or also by providing reaction conditions necessary for amplification, e.g. Setting a required reaction temperature for one or more process steps.
  • a component e.g. by adding a nucleic acid chain comprising a target sequence (e.g., a starting nucleic acid chain), or a polymerase or divalent metal ions, or also by providing reaction conditions necessary for amplification, e.g. Setting a required reaction temperature for one or more process steps.
  • the amplification can be carried out until the desired amount of nucleic acid to be amplified is reached.
  • the amplification reaction is carried out for a time which would have been sufficient in the presence of a nucleic acid to be amplified in order to obtain a sufficient amount.
  • the amplification reaction is carried out over a sufficient number of synthesis cycles (doubling times) which would have been sufficient in the presence of a nucleic acid to be amplified in order to obtain a sufficient amount.
  • the reaction can be stopped by various interventions. For example, by changing the temperature (e.g., cooling or heating, whereby, for example, polymerase is impaired in its function) or by adding a substance which stops a polymerase reaction, e.g. EDTA or formamide.
  • a temperature e.g., cooling or heating, whereby, for example, polymerase is impaired in its function
  • a substance which stops a polymerase reaction e.g. EDTA or formamide.
  • the amplified nucleic acid chain can be used for further analysis.
  • synthesized nucleic acid chains can be analyzed by various detection methods. For example, fluorescence-labeled oligonucleotide probes can be used or sequencing methods (Sanger sequencing or Next-Generation sequencing), solid-phase analyzes such as microarray or bead-array analyzes, etc.
  • the synthesized nucleic acid chain can be used as a substrate / template in further primer extension reactions become.
  • the progress of the synthesis reaction is monitored during the reaction. This can be done, for example, by using intercalating dyes, e.g. Sybrgreen or Evagreen, or using labeled primers (e.g., Lux primer or Scorpion primer) or using fluorescently labeled oligonucleotide probes.
  • intercalating dyes e.g. Sybrgreen or Evagreen
  • labeled primers e.g., Lux primer or Scorpion primer
  • fluorescently labeled oligonucleotide probes e.g., fluorescently labeled oligonucleotide probes.
  • the detection of the change in fluorescence during amplification is implemented in a detection step of the method.
  • the temperature and the duration of this step can be adapted to the respective requirements of the oligonucleotide probe.
  • the temperatures of the detection step include, for example, ranges between 20 ° C and 75 ° C, in particular between 40 and 70 ° C, in particular between 55 and 70 ° C.
  • the reaction is illuminated with light of a wavelength which is capable of excitation into a used fluorophore of the detection system (a donor or a fluorescent reporter).
  • the signal detection is usually parallel to excitation, whereby the specific fluorescence signal is detected and its intensity is quantified.
  • the amplification method can be used to verify the presence of a target nucleic acid chain in a biological material or diagnostic material as part of a diagnostic procedure.
  • reaction conditions of a PCR are well known to a person skilled in the art.
  • temperatures shown in FIGS. 3-11 include the following ranges:
  • T1 55 ° C (+/- 5 ° C)
  • T2 65 ° C (+/- 5 ° C)
  • T3 95 ° C (+/- 5 ° C)
  • T4 45 ° C (+/- 5 ° C)
  • T5 70 ° C (+/- 5 ° C)
  • temperatures shown in FIGS. 3-11 include the following ranges:
  • T1 55 ° C (+/- 10 ° C)
  • T2 65 ° C (+/- 10 ° C)
  • T3 95 ° C (+/- 10 ° C)
  • T4 45 ° C (+/- 10 ° C)
  • T5 70 ° C (+/- 10 ° C)
  • temperatures shown in FIGS. 3-11 include the following ranges:
  • T1 55 ° C (+/- 15 ° C)
  • T2 65 ° C (+/- 10 ° C)
  • T3 95 ° C (+/- 10 ° C)
  • T4 45 ° C (+/- 20 ° C)
  • T5 70 ° C (+/- 10 ° C)
  • the temperatures shown in FIGS. 3-11 include the following ranges:
  • T1 55 ° C (+/- 5 ° C)
  • T2 65 ° C (+/- 3 ° C)
  • T3 95 ° C (+/- 10 ° C)
  • T4 45 ° C (+/- 5 ° C)
  • T5 70 ° C (+/- 3 ° C)
  • the time of incubation during individual temperature steps may, in some embodiments, be between 0.1 sec and 60 min, in particular between 1 sec and 10 min, in particular between 1 sec and 5 min.
  • PCR cycles The cyclic temperature changes are often referred to as PCR cycles.
  • a PCR cycle involves a complete change in temperature from T1, T2 to T3.
  • the temperature ranges used are primarily intended to support the following steps of the second amplification:
  • T1, T2, T4 and T5 Temperatures Hybridization of the third and fourth primers to their complementary segments in the respective template strands and extension of a third and fourth primer extension product.
  • T3 temperature is used to separate double strands comprising at least one of the primer extension products. All double-stranded nucleic acid chains (eg double-stranded target sequences, first amplification fragments 1 .1, second amplification fragments 2. 1) and intermediates comprising P1 .1-ext and P4.1-Ext-part 1 or P2 and P3.1 -Ext part 1) converted by the action of temperature in the single-stranded form.
  • All double-stranded nucleic acid chains eg double-stranded target sequences, first amplification fragments 1 .1, second amplification fragments 2.
  • intermediates comprising P1 .1-ext and P4.1-Ext-part 1 or P2 and P3.1 -Ext part
  • T3 temperature may serve to activate hot-start forms of the second polymerases (e.g., templates inactivated by an antibody) or to inactivate a first polymerase.
  • thermolabile controller oligonucleotides can be cleaved by this temperature (T3).
  • thermoactivatable primers three and four can be activated.
  • reaction temperatures in combination with respective PCR primers can influence the production of products / formation of intermediates during the second amplification.
  • the amount of yields of individual products and intermediates depends on several factors. For example, higher concentrations of individual primers generally favor the yields of products / intermediates. Furthermore, the formation of products / intermediates can be influenced by the reaction temperature and the binding / affinity of individual reactants to each other (affinity of the binding of individual For example, longer oligonucleotides generally bind better than shorter oligonucleotides at higher temperatures, and CG content may also play a role in complementary sequence segments: CG-richer sequences also bind more strongly than AT-rich at higher temperatures sequences. Furthermore, modifications such as MGB or 2-amino-dA or LNA can increase the binding strength of primers to their respective complementary segments, which also results in preferential binding of primers at higher temperatures.
  • longer primers are used for the PCR (e.g., between 25 and 60 nucleotides, with a CG content of over 40%) having a relatively higher Tm with their complementary regions (e.g., 60-70 ° C).
  • This allows hybridization steps and primer extension steps of the second amplification to be performed at higher temperatures (e.g., T2 or T5).
  • the reaction in combination with relatively short first and second primers (from the first amplification), the reaction can be controlled to preferentially form primer extension products P4.1-Ext and P3.1-Ext and co-amplify primer extension products starting from P1 .1 and P2.1 during the PCR amplification can be largely suppressed.
  • third and fourth primers including, for example, only a short 3 ' segment capable of binding to the target sequence or its equivalents (eg, this segment is between 6 and 15 nucleotides in length)
  • this segment is between 6 and 15 nucleotides in length
  • PCR primers which comprise, for example, mismatches to only essentially complementary first amplification fragment 1 .1.
  • primer binding of the third and / or fourth primer to their complementary segments of the first amplification fragment occurs such that, despite a small length of the complementary segment and / or mismatch, the polymerase starting from these primers hybridized to individual strands of the first amplification fragment can perform a template-dependent primer extension and primer extension products comprising a third and fourth primer oligonucleotide can be synthesized.
  • the enzymatic synthesis also takes place in the second amplification generally in a buffered aqueous solution.
  • buffer solutions dissolved conventional buffer substances, such as Tris-HCl, Tris-acetate, potassium glutamate, HEPES buffer, sodium glutamate in conventional concentrations can be used.
  • the pH of these solutions is usually between 7 and 9.5, in particular about 8 to 8.5.
  • the buffer conditions can be adapted, for example, according to the recommendation of the manufacturer of the polymerase used.
  • Tm depressants e.g., DMSO, betaines, TPAC
  • Tm depressors e.g., DMSO, betaines, TPAC
  • Tween 20 or T riton 100 can also be added in conventional amounts to the buffer.
  • EDTA or EGTA can be added to complex heavy metals in conventional amounts.
  • Polymerase stabilizing substances such as trehalose or PEG 6000 may also be added to the reaction mixture.
  • the nucleic acid chain used or to be used at the beginning of an amplification reaction can be referred to as a starting nucleic acid chain (FIG. 1).
  • a starting nucleic acid chain comprises a target sequence.
  • primer extension reactions By binding primers to their corresponding primer binding sites (PBS 1 and PBS 2) and initiating appropriate primer extension reactions, this is done by generating first primer extension products. These are synthesized as specific copies of the nucleic acid chain present at the beginning of the reaction.
  • this nucleic acid chain (starting nucleic acid chain) to be used in the reaction mixture prior to the beginning of the amplification reaction may be identical to the nucleic acid chain to be amplified.
  • the amplification reaction only increases the amount of such a nucleic acid chain.
  • the nucleic acid to be amplified and the starting nucleic acid chain differ in that the starting nucleic acid chain differs from the arrangement individual sequence elements of the nucleic acid sequence to be amplified, but the sequence composition of the starting nucleic acid chain may differ from the sequence of the nucleic acid chain to be amplified.
  • new sequence contents based on starting nucleic acid chain
  • sequence elements of a nucleic acid chain to be amplified may differ from such sequence elements of a starting nucleic acid chain in their sequence composition (eg primer binding sites or primer sequences).
  • the starting nucleic acid serves only as an initial template for the specific synthesis of the nucleic acid chain to be amplified. This initial template may remain in the reaction mixture until the end of the amplification. Due to the exponential nature of the amplification, however, the amount of nucleic acid chain to be amplified at the end of an amplification reaction is greater than the amount of starting nucleic acid chain added to the reaction.
  • the startup nucleic acid chain may comprise at least one sequence segment which is not amplified. Such a start nucleic acid chain is thus not identical to the sequence to be amplified. Such sections which are not to be amplified may represent, for example as a consequence of sequence preparation steps or as a consequence of previous sequence manipulation steps, a sequence section of a starting nucleic acid chain.
  • the starting nucleic acid sequence to be inserted into the reaction mixture prior to initiation of the reaction includes at least one target sequence.
  • such a starting nucleic acid chain includes at least one target sequence and still other non-target sequence sequences.
  • sequence segments comprising the target sequence are multiplied exponentially and other sequence segments are either not or only partially exponentially propagated.
  • An example of such a start nucleic acid chain is a nucleic acid sequence which includes a target sequence and which comprises a Sequence Fragment-A and comprises a Sequence Fragment-B.
  • Sequence fragment-A of the starting nucleic acid chain comprises a sequence which has significant homology with the sequence of one of the two primers used in the amplification or is substantially identical to the copiable portion of the 3 ' segment of the one primer. Upon synthesis of a complementary strand to this segment, a complementary sequence is generated which represents a corresponding primer binding site.
  • sequence fragment B of the starting nucleic acid chain comprises a sequence which is complementary to the binding of a corresponding further primer or its 3 ' segment is suitable for forming an extension-capable primer-template complex, wherein sequence fragment A and sequence fragment B are predominantly non-complementary in relation to each other.
  • the reaction mixture of an amplification process is given a starting nucleic acid chain which has the following properties:
  • sequence fragment-A is located in the 5 ' segment of the starting nucleic acid chain.
  • this sequence fragment-A forms a restriction of the nucleic acid chain strand in the 5 ' direction.
  • sequence fragment-B lies in the 3 'direction, relative to the binding site of the first primer and relative to the polarity of the first primer ("downstream"), from sequence fragment-A.
  • this sequence fragment-B forms a restriction of the nucleic acid chain strand in the 3 ' direction.
  • this sequence fragment-B does not represent a 3 ' -chain boundary of the nucleic acid chain strand, but is flanked by further sequences from the 3 ' side.
  • these sequences are not targeting sequences and do not participate in exponential amplification.
  • the target sequence comprises at least one of either Sequence Fragment A or Sequence Fragment B. In certain embodiments, the target sequence is between sequence fragment A and sequence fragment B.
  • such a starting nucleic acid chain can serve as a template for the synthesis of a first primer extension product.
  • the starting nucleic acid chain can be provided, for example, in the context of a primer extension reaction using the second primer oligonucleotide as a sequence segment of a longer starting nucleic acid chain during a preparatory step before the exponential amplification and converted into a single-stranded form.
  • the starting nucleic acid chain may be, for example, a genomic DNA or RNA and serves as a source of the target sequence.
  • the start nucleic acid chain comprises in the 5 ' segment of the nucleic acid chain a segment 1 comprising sequence sections of the second primer oligonucleotide and limiting in the 5 ' direction, a segment 2 comprising a target sequence or its parts, a segment 3, which comprises a primer binding site for the first primer oligonucleotide and a segment 4 which locates a non-target sequence in the 3 ' segment of the starting nucleic acid chain and thus flanks segment 3 on the 3 ' side.
  • the first primer can bind to such a starting nucleic acid chain in segment 3 at least with the 3 ' segment of its first region and can be correspondingly extended in the presence of a polymerase and nucleotides. This results in a first primer extension product which is complementary to the template strand of the starting nucleic acid chain and is limited in its length.
  • a such primer extension product via the oligonucleotide controller are separated from its template strand sequence-specific, so that corresponding sequence sections in the 3 ' segment of the now synthesized first primer extension product are available as a binding site for the second primer oligonucleotide.
  • a starting nucleic acid chain thus comprises the following sequence fragments (FIG. 50):
  • Sequence segment-1 (labeled segment 1) which comprises a sequence which has significant homology with the sequence of the second primer or is substantially identical to the copiable portion of the 3 ' segment of the second primer. This sequence segment-1 is located in the 5 ' segment of the starting nucleic acid chain, in particular this sequence segment-1 forms a boundary of the nucleic acid chain strand in the 5 ' direction.
  • sequence segment-3 (labeled segment 3), which comprises a sequence which is suitable for complementary binding of the first region of the first primer or its 3 ' segment to form an extension-capable primer-template complex.
  • sequence segment-3 is in the 3 'direction, ("upstream") of the sequence segment-1.
  • Target sequence which lies partially or completely between segment 1 and segment 3 (this portion of the target sequence is referred to as segment 2).
  • the target sequence comprises segment 2, as well as at least one of segment 1 and / or segment 3.
  • the start nucleic acid chain in the 3 ' segment comprises flanking sequence segments (termed segment 4) which are not amplified.
  • a starting nucleic acid chain may serve as a template for the synthesis of a second primer extension product.
  • the starting nucleic acid chain can be provided, for example, in the context of a primer extension reaction using the first primer oligonucleotide as a sequence segment of a longer starting nucleic acid chain during a preparatory step before the exponential amplification and converted into a single-stranded form.
  • the starting nucleic acid chain may be, for example, a genomic DNA or RNA and serves as the source of the target sequence (shown schematically as a double strand).
  • the start nucleic acid chain comprises in the 5 ' segment of the nucleic acid chain a segment 5 comprising sequence segments of the first primer oligonucleotide and limiting in the 5 ' direction, a segment 6 comprising a target sequence or its moieties, a segment 7, what comprises a primer binding site for the second primer oligonucleotide and a segment 8 which locates a non-target sequence in the 3 ' segment of the starting nucleic acid chain and thus flanks segment 7 on the 3 ' side.
  • the second primer can bind at least to the 3 ' segment of its first region to such a starting nucleic acid chain in segment 7 and in the presence of a polymerase and nucleotides correspondingly up to the stop segment of the first primer oligonucleotide be extended. This results in a second primer extension product which is complementary to the template strand of the starting nucleic acid chain and is limited in its length.
  • such a primer extension product can be detached from its template strand sequence-specific via the controller oligonucleotide so that corresponding sequence segments in the 3 ' segment of the now synthesized second primer extension product are available as a binding site for the first primer oligonucleotide.
  • a start nucleic acid chain comprises the following sequence fragments ( Figure 51):
  • segment 5 comprising a sequence having significant homology with the sequence of the first region of the first primer or being substantially identical to the copiable portion of the first region of the first primer.
  • This segment-5 lies in the 5 ' segment of the starting nucleic acid chain and is flanked by a non-copyable oligonucleotide tail (analogous to the first primer oligonucleotide), in particular this segment forms a boundary of the copiable nucleic acid chain strand in 5 ' .
  • a non-copyable oligonucleotide tail analogous to the first primer oligonucleotide
  • segment 7 The sequence fragment-7 (called segment 7), which comprises a sequence which is suitable for the complementary binding of a second primer or its 3 ' segment to form an extension-capable primer-template complex.
  • the segment 7 lies in the 3 'direction ("upstream") of the segment 5.
  • Target sequence which lies partially or completely between segment 5 and segment 7 (this portion of the target sequence is referred to as segment 6).
  • the target sequence comprises segment 6 and at least one of segment 5 and / or segment 7.
  • the starting nucleic acid chain includes flanking 3 ' segments
  • the starting nucleic acid chain serves as a template for the initial generation of respective primer extension products. It thus represents the starting template for the nucleic acid chain to be amplified.
  • the starting nucleic acid chain does not necessarily have to be identical to the nucleic acid chain to be amplified.
  • non-denaturing reaction conditions are maintained for a duplex during the exponential amplification process. Therefore, it is advantageous if the starting nucleic acid chain has a restriction in its polymerase-extendable 5 ' sequence segment, resulting in a stop in the enzymatic extension of a corresponding primer. This limits the length of the primer extension fragments generated under reaction conditions. This can have an advantageous effect on the strand displacement by the controller oligonucleotide and lead to a dissociation of the respective strand, so that primer binding sites are converted into the single-stranded state and thus become accessible for a recapture of primers.
  • the first primer oligonucleotide (primer-1) is a nucleic acid chain which includes at least the following regions ( Figures 12-14):
  • a first primer region in the 3 ' segment of the first primer oligonucleotide capable of substantially sequence-specific binding to a strand of nucleic acid chain to be amplified
  • a second region, coupled directly or via a linker, to the 5 ' end of the first primer region of the first primer oligonucleotide comprising a polynucleotide tail capable of binding a controller oligonucleotide and promoting strand displacement step c). by controller oligonucleotide, wherein the polynucleotide tail remains substantially single-stranded under reaction conditions, ie does not form a stable hairpin structure or ds-structures, and in particular is not copied by the polymerase.
  • the total length of the first primer oligonucleotide is between 10 and 80, more preferably between 15 and 50, more preferably between 20 and 30 nucleotides or their equivalents (e.g., nucleotide modifications).
  • the structure of the first primer oligonucleotide is adapted to undergo reversible binding to the controller oligonucleotide under selected reaction conditions. Furthermore, the structure of the first primer oligonucleotide is adapted to its primer function. Furthermore, the structure is adapted so that a strand displacement can be performed by means of controller oligonucleotide. Overall, structures of the first and second regions are matched to each other so that exponential amplification can be performed.
  • the first and the second region of the primer are coupled in a conventional 5 ' -3 ' arrangement.
  • the coupling of both sections takes place via a 5 ' -5 ' bond, so that the second region has a reverse direction than the first region.
  • the coupling between the first and second regions is a conventional DNA for 5'-3 '-Phosphodiester coupling. In certain embodiments, it is a 5 '-5' -Phosphodiester coupling. In certain embodiments, it is a 5 '-3' - phosphodiester linkage, wherein between adjacent terminal nucleotides or nucleotide modifications of the two regions at least one linker (eg, a C3, C6, C12 or HEG linker or abasic modification) is positioned.
  • linker eg, a C3, C6, C12 or HEG linker or abasic modification
  • nucleotide modifications may include different nucleotide modifications.
  • individual elements of nucleotides can be modified: nucleobase and backbone (sugar content and / or phosphate content).
  • modifications may be used which lack or are modified at least one component of the standard nucleotide building blocks, e.g. PNA.
  • a second portion of the first primer oligonucleotide includes additional sequences that do not bind to the controller oligonucleotide. These sequences can be used for other purposes, such as binding to the solid phase. These sequences are located in particular at the 5 ' end of the polynucleotide tail.
  • a first primer oligonucleotide may include characteristic label.
  • characteristic label are dyes (e.g., FAM, TAMRA, Cy3, Alexa 488, etc.) or biotin or other groups which can be specifically bound, e.g. Digoxigenin.
  • the sequence length is between about 3-30 nucleotides, in particular between 5 and 20 nucleotides, the sequence being predominantly complementary to the 3 ' segment of a strand of the nucleic acid chain to be amplified.
  • this primer region must be able to specifically bind to the complementary 3 ' segment of a second primer extension product.
  • This first area should be copied in reverse synthesis and also serves as a template for 2nd strand.
  • the nucleotide building blocks are in particular linked to each other via conventional 5 'to 3' Phosphodie bond or Phosphothioester bond.
  • the first primer region preferably includes nucleotide monomers which do not or only insignificantly affect the function of the polymerase, for example:
  • the first primer region serves as the initiator of the synthesis of the first primer extension product in the amplification.
  • the first region comprises at least one phosphorothioate compound, so that no degradation of the 3 ' end of the primer by 3 ' exonuclease activity of polymerases can take place.
  • sequence of the first region of the first primer oligonucleotide and the sequence of the second region of the controller oligonucleotide are in particular complementary to one another.
  • the first primer region or its 3 ' segment can bind to sequence segments of a target sequence.
  • the second region of the first primer oligonucleotide is a nucleic acid sequence comprising at least one polynucleotide tail, which in particular remains uncoupled from the polymerase during the synthesis reaction and which is capable of binding to the first region of the controller oligonucleotide.
  • the segment of the second region, which predominantly undergoes this binding with the controller oligonucleotide, may be referred to as the polynucleotide tail.
  • the second region of the first primer oligonucleotide must not only specifically bind the controller oligonucleotide under reaction conditions, but also participate in the process of strand displacement by means of controller oligonucleotide.
  • the structure of the second region must therefore be suitable for bringing about spatial proximity between the controller oligonucleotide and the corresponding duplex end (in particular, the 3 ' end of the second primer extension product).
  • the design of the structure of the second region of the first primer oligonucleotide is shown in more detail in several embodiments. In this case, the arrangement of the oligonucleotide segments and used modifications are taken into account, which lead to a stop in the polymerase-catalyzed synthesis.
  • the length of the second region is between 3 and 60, in particular between 5 and 40, in particular between 6 and 15 nucleotides or their equivalents.
  • the sequence of the second area may be chosen arbitrarily. In particular, it is not complementary with the nucleic acid to be amplified and / or with the second primer oligonucleotide and / or with the first region of the first primer oligonucleotide. Furthermore, it contains in particular no self-complementary segments, such as hairpins or Stemmloops.
  • the sequence of the second region is particularly matched to the sequence of the first region of the controller oligonucleotide, so that both sequences can bind under reaction conditions.
  • this bond is under Reaction conditions reversible: there is thus a balance between components bound together and unbound components.
  • the sequence of the second region of the first primer oligonucleotide is chosen in particular such that the number of complementary bases which can bind to the first region of the controller oligonucleotide is between 1 and 40, in particular between 3 and 20, in particular between 6 and 15 lies.
  • the function of the second area is, inter alia, the binding of the controller oligonucleotide. In certain embodiments, this binding is particularly specific so that a second region of a first primer oligonucleotide can bind a specific controller oligonucleotide. In another embodiment, a second region may bind more than one controller oligonucleotide under reaction conditions.
  • the degree of complementarity between the second region of the first primer oligonucleotide and the first region of the controller oligonucleotide may be between 20% and 100%, in particular between 50% and 100%, in particular between 80% and 100%.
  • the respective complementary regions may be positioned immediately adjacent to each other or may comprise non-complementary sequence segments therebetween.
  • the second region of the first primer oligonucleotide may, in certain embodiments, include at least one Tm modifying modification.
  • Tm enhancing modifications nucleotide modifications or non-nucleotide modifications
  • Tm-lowering modifications may also be used, such as inosine nucleotide.
  • linkers e.g., C3, C6, HEG linkers
  • the controller oligonucleotide For strand displacement, the controller oligonucleotide must be placed in close proximity to the double-stranded end of the nucleic acid to be amplified.
  • This double-stranded end consists of segments of the first primer region of the first primer extension product and a correspondingly complementary thereto 3 ' segment of the second primer extension product.
  • the polynucleotide tail predominantly complements the controller oligonucleotide under reaction conditions, thereby causing a transient approach of the second region of the controller oligonucleotide and the first region of an extended primer. Extension product, so that a complementary bond between these elements can be initiated as part of a strand displacement process.
  • binding of the controller oligonucleotide to the polynucleotide tail of the first primer oligonucleotide immediately results in such contact.
  • the polynucleotide tail and the first primer region of the first primer oligonucleotide must be directly coupled to each other. Thanks to such an arrangement, after a binding of a controller oligonucleotide in its first region, contact can be made directly between complementary bases of the second region of the controller oligonucleotide and corresponding bases of the first primer region, so that strand displacement can be initiated.
  • structures of the second region of the first primer oligonucleotide are located between structures of the polynucleotide tail and the first primer region. After binding of a controller oligonucleotide to the polynucleotide tail, it is thus not positioned directly at the first primer region, but at a certain distance from it.
  • the structures between the uncopiable polynucleotide tail and the copiable first primer region of the primer oligonucleotide can generate such a spacing.
  • This distance has a value which is between 0.1 and 20 nm, in particular between 0.1 and 5 nm, in particular between 0.1 and 1 nm.
  • Such structures are, for example, linkers (e.g., C3 or C6 or HEG linkers) or non-controller oligonucleotide complementary segments (e.g., in the form of non-complementary, non-copyable nucleotide modifications).
  • the length of these structures can generally be measured in chain atoms. This length is between 1 and 200 atoms, in particular between 1 and 50 chain atoms, preferably between 1 and 10 chain atoms.
  • the second region of the first primer oligonucleotide generally comprises sequence structures which cause the polymerase to stop in the synthesis of the second primer extension product after the polymerase releases the polymerase first primer area has been successfully copied. These structures are said to prevent copying of the polynucleotide tail of the second region. The polynucleotide tail thus remains uncoated, in particular, by the polymerase.
  • such structures are between the first primer region and the polynucleotide tail.
  • the sequence of the polynucleotide tail may include nucleotide modifications that result in the termination of the polymerase.
  • a sequence segment of the second region of the first primer oligonucleotide may comprise both functions: it is both a polynucleotide tail and a polymerase-terminating sequence of nucleotide modifications. Modifications in the second region of the first primer oligonucleotide which result in a synthetic stop and thus leave the polynucleotide tail uncopyable are summarized in this application by the term "first blocking moiety or a stop region".
  • oligonucleotide synthesis Several building blocks in oligonucleotide synthesis are known which prevent the polymerase from reading the template and lead to the termination of polymerase synthesis.
  • non-copyable nucleotide modifications or non-nucleotide modifications are known.
  • There are also synthetic types / arrangements of nucleotide monomers within an oligonucleotide which result in the termination of the polymerase eg, 5 'to 5 ' or 3 'to 3 ' ).
  • Primer oligonucleotides with a non-copyable polynucleotide tail are also known in the art (eg Scorpion primer structures or primers for binding to the solid phase).
  • Both primer variants describe primer oligonucleotide structures which are able to initiate the synthesis of a strand on the one hand so that a primer extension reaction can take place.
  • the result is a first strand, which also integrates the primer structure with tail in the primer extension product.
  • the second strand is extended to the "blocking moiety / stop structure" of the primer structure.
  • Both described primer structures are designed in such a way that the 5 ' portion of the primer oligonucleotide remains single-stranded and is not copied by the polymerase.
  • the second region of the primer oligonucleotide comprises a polynucleotide tail that has a conventional 5 ' to 3 ' array in its entire length and includes non-copyable nucleotide modifications.
  • non-copyable nucleotide modifications include, for example, 2 '-0-alkyl-RNA modifications, PNA, morpholino. These modifications may be distributed differently in the second primer region.
  • the proportion of non-copyable nucleotide modifications may be between 20% and 100% in the polynucleotide tail, in particular more than 50% of the nucleotide building blocks.
  • these nucleotide modifications are in the 3 ' segment of the second region and thus adjacent to the first region of the first primer oligonucleotide.
  • sequence of non-copyable nucleotide modifications is at least partially complementary to the sequence in the template strand such that primer binding to the template occurs involving at least a portion of these nucleotide modifications. In certain embodiments, the sequence of non-copyable nucleotide modifications is non-complementary to the sequence in the template strand.
  • the non-copyable nucleotide modifications are in particular covalently coupled to each other and thus represent a sequence segment in the second region.
  • the length of this Segment comprises between 1 and 40, in particular between 1 and 20 nucleotide modifications, in particular between 3 and 10 nucleotide modifications.
  • the second region of the first primer oligonucleotide comprising a polynucleotide tail, which 'has and non-copyable nucleotide modifications include (for example 2' in its entire length, a conventional arrangement of 5'-3 -0-alkyl modifications ) and at least one non-nucleotide linker (eg, C3, C6, HEG linker).
  • a non-nucleotide linker has the function of covalently linking adjacent nucleotides or nucleotide modifications while at the same time site-specifically disrupting the synthesis function of the polymerase.
  • non-nucleotide linker should not remove the structures of the polynucleotide tail and the first primer region too far apart. Rather, the polynucleotide tail should be in close proximity to the first primer region.
  • a non-nucleotide linker is taken to mean modifications which do not have a length of more than 200 chain atoms, in particular not more than 50 chain atoms, in particular not more than 10 chain atoms. The minimum length of such a linker can be one atom.
  • An example of such non-nucleotide linkers are straight or branched alkyl linkers with an alkyl chain which include at least one carbon atom, more preferably at least 2 to 30, especially 4 to 18.
  • Such linkers are in oligonucleotide chemistry are well known (eg, C3, C6, or C12 linkers) and can be introduced during solid phase synthesis of oligonucleotides between the sequence of the polynucleotide tail and the sequence of the first region of the first primer oligonucleotide.
  • Another example of such non-nucleotide linkers is linear or branched polyethylene glycol derivatives.
  • a well-known example in oligonucleotide chemistry is hexaethylene glycol (HEG).
  • HEG hexaethylene glycol
  • Another example of such non-nucleotide linkers are abasic modifications (eg THF modification, as analog of d ribose).
  • modifications are integrated into a second region, they can effectively interfere with a polymerase in its copying function during its synthesis of the second primer extension product, such that segments located in the 3 'direction remain uncopied after such modification.
  • the number of such modifications in the second range can be between 1 and 100, in particular between 1 and 10, in particular between 1 and 3.
  • the position of such a non-nucleotide linker may be at the 3 ' end of the second region, thus representing the transition to the first region and the second region of the primer oligonucleotide.
  • the location of the non-nucleotide linker in the middle segment of the second region may also be used.
  • the 3 ' segment of the polynucleotide tail includes at least one, in particular several, eg between 2 and 20, in particular between 2 and 10 non-limiting copyable nucleotide modifications. These non-copyable nucleotide modifications are in particular at the transition between the first and the second region of the primer oligonucleotide.
  • the second region of the primer oligonucleotide comprises a polynucleotide tail having in its entire length an array of 5 'to 3 ' - and at least one nucleotide monomer in a "reverse" array of 3 'to 5 '- includes and which are positioned at the transition between the first and the second region of the first primer oligonucleotide.
  • the second region of the primer oligonucleotide comprises a polynucleotide tail, such polynucleotide tail consisting entirely of nucleotides which are directly adjacent to the first region of the first primer oligonucleotide in reverse order such that the coupling of the first and second nucleotides of the second area through 5 ' - 5 ' position.
  • the 3 ' -terminal nucleotide of the polynucleotide tail should be blocked at its 3 ' OH end to prevent side reactions.
  • a terminal nucleotide can be used which has no 3 ' -OH group, for example a dideoxy nucleotide.
  • the corresponding nucleotide arrangement is to be adapted in the controller oligonucleotide.
  • the first and the second portion of the controller oligonucleotide in 3 'to 3' arrangement have to be linked.
  • the second region of the primer oligonucleotide comprises a polynucleotide tail having a conventional 5 ' to 3 ' configuration in its entire length and including at least one nucleotide modification which is not a complementary nucleobase for the polymerase, when the synthesis is performed with all natural dNTP (dATP, dCTP, dGTP, dTTP or dUTP).
  • dNTP dATP, dCTP, dGTP, dTTP or dUTP
  • iso-dG or iso-dC nucleotide modifications can be integrated as single, but in particular more (at least 2 to 20) nucleotide modifications in the second region of the first primer oligonucleotide.
  • nucleobase modifications are various modifications of the extended genetic alphabet.
  • such nucleotide modifications do not support complementary base pairing with natural nucleotides such that a polymerase (at least theoretically) does not contain a nucleotide from the series (dATP, dCTP, dGTP, dTTP or dUTP). In reality, rudimentary incorporation may still occur, especially at higher concentrations of dNTP substrates and prolonged incubation times (eg, 60 minutes or longer).
  • nucleotide modifications positioned at adjacent sites are used.
  • the stop of polymerase synthesis is effected by the lack of suitable complementary substrates for these modifications.
  • Oligonucleotides with iso-dC or iso-dG can be synthesized by standard methods and are available from several commercial suppliers (eg Trilink Technologies, Eurogentec, Biomers GmbH).
  • the sequence of the first region of the controller oligonucleotide can also be adapted to the sequence of such a second primer region.
  • complementary nucleobases of the extended genetic alphabet can be integrated into the first region of the controller oligonucleotide accordingly during the chemical synthesis.
  • iso-dG may be integrated in the second region of the first primer nucleotide, its complementary nucleotide (iso-dC-5-Me) may be placed at the appropriate location in the first region of the controller oligonucleotide.
  • the termination of the synthesis of polymerase in the second region can be achieved in various ways.
  • this blockade preferably takes place only when the polymerase has copied the first region of the first primer oligonucleotide. This ensures that a second primer extension product has an appropriate primer binding site in its 3 ' segment. This primer binding site is exposed as part of the strand displacement and is thus available for a new binding of another first primer oligonucleotide.
  • the primer extension reaction remains in front of the polynucleotide tail. Because this polynucleotide tail remains single stranded for interaction with the controller oligonucleotide and thus is available for binding, it promotes the initiation of the strand displacement reaction by the controller oligonucleotide by placing it in the immediate vicinity of the corresponding complementary segments of the controller oligonucleotide Near the matching duplex-end brings. The distance between the complementary part of the controller oligonucleotide (second region) and the complementary part of the extended primer oligonucleotide (first region) is thereby reduced to a minimum. Such spatial proximity facilitates the initiation of strand displacement.
  • a complementary sequence of controller oligonucleotide is now in the immediate vicinity of the appropriate duplex end. This results in competition for binding to the first region of the first primer oligonucleotide between the strand of the controller oligonucleotide and the template strand complementary to the primer.
  • the initiation of the nucleic acid-mediated strand displacement process occurs.
  • distances between the 5 ' segment of the first primer region of the first primer oligonucleotide, which binds to a complementary strand of the template and forms a complementary duplex, and a correspondingly complementary sequence segment in the controller oligonucleotide, when bound to polynucleotide Tail of the second region of the first primer oligonucleotide in the following ranges are: between 0.1 and 20 nm, in particular between 0.1 and 5 nm, in particular between 0.1 and 1 nm. In this particular case, this distance is less than 1 nm.
  • this distance corresponds to a distance of less than 200 atoms, in particular less than 50 atoms, in particular less than 10 atoms. In the special case, this distance is an atom.
  • the distance information is for guidance only and illustrates that shorter distances between these structures are preferred. The measurement of this distance is in many cases only possible by analyzing the exact structures of oligonucleotides and measuring the measurement of sequence distances or of linker lengths.
  • the first primer may also include additional sequence segments that are not required to interact with the controller oligonucleotide or template strand. Such sequence segments may, for example, bind further oligonucleotides which are used as detection probes or immobilization partners when bound to the solid phase.
  • the first primer oligonucleotide can be used in several partial steps. First and foremost, it performs a primer function in the amplification.
  • the primer extension reaction is carried out using the second primer extension product as a template.
  • the first primer oligonucleotide may use the starting nucleic acid chain as a template at the beginning of the amplification reaction.
  • the first primer oligonucleotide may be used in the preparation / provision of a starting nucleic acid chain.
  • the first primer serves as the initiator of the synthesis of the first primer extension product using the second primer extension product as a template.
  • the 3 ' segment of the first primer comprises a sequence which can bind predominantly complementary to the second primer extension product.
  • Enzymatic extension of the first primer oligonucleotide using the second primer extension product as template results in formation of the first primer extension product.
  • a first primer extension product comprises the target sequence or its sequence parts.
  • the sequence of the copyable portion of the first primer oligonucleotide is recognized by the polymerase as a template and a corresponding complementary sequence is synthesized, so that a corresponding primer binding site for the first primer oligonucleotide results.
  • the synthesis of the first primer extension product occurs up to and including the 5 ' segment of the second primer oligonucleotide.
  • this product is bound to the second primer extension product to form a double-stranded complex.
  • the second primer extension product is displaced sequence-specifically from this complex by the controller oligonucleotide.
  • the controller oligonucleotide binds to the first primer extension product.
  • the second primer extension product may itself serve as a template for the synthesis of the first primer extension product.
  • the now vacated 3 ' segment of the first primer extension product can bind another second primer oligonucleotide so that a new synthesis of the second primer extension product can be initiated.
  • the first primer oligonucleotide can serve as the initiator of the synthesis of the first primer extension product starting from the starting nucleic acid chain at the beginning of the amplification.
  • the sequence of the first primer is completely complementary to the corresponding sequence segment of a starting nucleic acid chain.
  • the sequence of the first primer oligonucleotide is only partially complementary to the corresponding sequence segment of a starting nucleic acid chain.
  • this divergent complementarity is not intended to prevent the first primer oligonucleotide from starting a predominantly sequence-specific primer extension reaction.
  • the respective differences in complementarity of the first primer oligonucleotide to the respective position in the starting nucleic acid chain are in particular in the 5 ' segment of the first region of the first primer oligonucleotide, so that in the 3 ' segment predominantly complementary base pairing and initiation of the synthesis is possible .
  • the first 4-10 positions in the 3 ' segment should be fully complementary to the template (starting nucleic acid chain).
  • the remaining nucleotide positions may differ from a perfect complementarity.
  • the extent of perfect complementarity in the remaining 5 ' segment of the first region of the first primer oligonucleotide can range between 50% to 100%, more preferably between 80% and 100%, of the base composition.
  • the first primer oligonucleotide may thus initiate synthesis of a starting nucleic acid chain.
  • replicable sequence segments of the first primer oligonucleotide are copied from the polymerase such that, in subsequent synthesis cycles, a fully complementary primer binding site within the second primer extension product for binding of the first Primer oligonucleotide is formed and is available in subsequent synthesis cycles.
  • the first primer oligonucleotide may be used in the preparation of a starting nucleic acid chain.
  • a first primer oligonucleotide can bind to a nucleic acid (for example a single-stranded genomic DNA or RNA or its equivalents comprising a target sequence) predominantly sequence-specific and initiate a template-dependent primer extension reaction in the presence of a polymerase.
  • the binding position is chosen such that the primer extension product comprises a desired target sequence.
  • the extension of the first primer oligonucleotide results in a nucleic acid strand which has a sequence complementary to the template.
  • Such a strand can be detached from the template (eg by heat or alkali) and thus converted into a single-stranded form.
  • a single-stranded nucleic acid chain can serve as a starting nucleic acid chain at the beginning of the amplification.
  • Such a start nucleic acid chain comprises in its 5 ' segment the sequence portions of the first primer oligonucleotide, furthermore it comprises a target sequence or its equivalents and a primer binding site for the second primer oligonucleotide. Further steps are explained in the section "Starting nucleic acid chain".
  • the synthesis of the first primer extension product is a primer extension reaction and forms a partial step in the amplification.
  • the reaction conditions during this step are adjusted accordingly.
  • the reaction temperature and the reaction time are chosen so that the reaction can take place successfully.
  • the particular preferred temperature in this step depends on the polymerase used and the binding strength of the respective first primer oligonucleotide to its primer binding site and includes, for example, ranges from 15 ° C to 75 ° C, especially from 20 to 65 ° C, in particular from 25 ° C to 65 ° C.
  • the concentration of the first primer oligonucleotide comprises ranges from 0.01 pmol / l to 50 pmol / l, in particular from 0.1 pmol / l to 20 pmol / l, in particular from 0.1 pmol / l to 10 pmol / l.
  • all steps of the amplification proceed under stringent conditions that prevent or slow down the formation of nonspecific products / by-products.
  • stringent conditions include, for example, higher temperatures, in particular above 50 ° C.
  • sequence-specific primer oligonucleotides are preferably used in each case for the amplification of corresponding respective target sequences.
  • sequences of the first, second primer oligonucleotide and the controller oligonucleotide are matched to one another such that side reactions, eg primer-dimer formation, are minimized.
  • the sequence of the first and second primer oligonucleotides are adapted to one another such that both primer oligonucleotides are incapable of undergoing an amplification reaction in the absence of one suitable template and / or a target sequence and / or a start nucleic acid chain to start or support.
  • the second primer oligonucleotide does not comprise a primer binding site for the first primer oligonucleotide and the first primer oligonucleotide does not comprise a primer binding site for the second primer oligonucleotide.
  • the primer sequences comprise extended self-complementary structures (self-complement).
  • the synthesis of the first and second primer extension products proceeds at the same temperature. In certain embodiments, the synthesis of the first and second primer extension products proceeds at different temperatures. In certain embodiments, synthesis of the first primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature. In certain embodiments, synthesis of the first primer extension product and strand displacement by controller oligonucleotide proceeds at different temperatures.
  • a controller oligonucleotide ( Figures 19-26) comprises:
  • a first single-stranded region which can bind to the polynucleotide tail of the second region of the first primer oligonucleotide
  • a second single-stranded region which can bind to the first region of the first primer oligonucleotide essentially complementary
  • a third single-stranded region which is substantially complementary to at least one segment of the extension product of the first primer extension product
  • controller oligonucleotide does not serve as a template for primer extension of the first or second primer oligonucleotide.
  • the sequence of the third region of the controller oligonucleotide is adapted to the sequence of the nucleic acid to be amplified, since this is a template for the order of the nucleotides in the extension product of a first primer.
  • the sequence of the second region of the controller oligonucleotide is adapted to the sequence of the first primer region.
  • the structure of the first region of the controller oligonucleotide is adapted to the sequence of the second region of the first primer oligonucleotide, especially the nature of the polynucleotide tail.
  • a controller oligonucleotide may also include other sequence segments that do not belong to the first, second or third region. For example, these sequences can be attached as flanking sequences at the 3 ' and 5 ' ends. In particular, these sequence segments do not interfere with the function of the controller oligonucleotide.
  • the structure of the controller oligonucleotide has in particular the following properties:
  • the individual areas are covalently bonded to each other.
  • the binding can be done for example via conventional 5 ' -3 ' bond.
  • a phospho-diester bond or a nuclease-resistant phospho-thioester bond can be used.
  • a controller oligonucleotide may bind by its first region to the polynucleotide tail of the first primer oligonucleotide, which binding is mediated primarily by hybridization of complementary bases.
  • the length of this first region is 3 to 80 nucleotides, in particular 4 to 40 nucleotides, in particular 6 to 20 nucleotides.
  • the degree of sequence match between the sequence of the first region of the controller oligonucleotide and the sequence of the second region of the first primer oligonucleotide may be between 20% and 100%, in particular between 50% and 100%, in particular between 80% and 100%.
  • binding of the first region of the controller oligonucleotide should be specific to the second region of the first primer oligonucleotide under reaction conditions.
  • the sequence of the first region of the controller oligonucleotide is chosen in particular such that the number of complementary bases that can bind to the second region of the first primer oligonucleotide complementary between 1 and 40, in particular between 3 and 20, in particular between 6 and 15 lies.
  • controller oligonucleotide since the controller oligonucleotide is not a template for the polymerase, it may include nucleotide modifications that do not support polymerase function, which may be both base modifications and / or sugar-phosphate backbone modifications.
  • the controller oligonucleotide may include, for example, in its first region, nucleotides and / or nucleotide modifications selected from the following list: DNA, RNA, LNA ("locked nucleic acids” analogues having 2 ' -4 ' bridge linkage in the sugar moiety), UNA ( "unlocked Nucleic acids” without binding between 2 '-3' -atoms of the sugar moiety), PNA ( "peptide nucleic acids” analogs), PTO (phosphorothioate), morpholino analogs, 2 '-0-alkyl-RNA modifications (such as 2 '-OMe, 2' -0 propargyl, 2 '-0- (2-methoxyethyl), 2'
  • nucleotides or Nucleotide modifications are linked together, for example, by conventional 5 ' -3 ' bonding or 5 ' -2 ' bonding.
  • a phospho-diester bond or a nuclease-resistant phospho-thioester bond can be used.
  • the controller oligonucleotide may include nucleotides and / or nucleotide modifications in its first region, the nucleobases being selected from the following list: adenines and their analogs, guanines and its analogs, cytosines and its analogs, uracil and its analogs, thymines and its analogues, inosine or other universal bases (eg nitroindole), 2-amino-adenine and its analogues, iso-cytosines and its analogues, iso-guanines and its analogues.
  • nucleobases being selected from the following list: adenines and their analogs, guanines and its analogs, cytosines and its analogs, uracil and its analogs, thymines and its analogues, inosine or other universal bases (eg nitroindole), 2-amino-adenine and its analogues, iso-cytosines and
  • the controller oligonucleotide may include in its first region non-nucleotide compounds selected from the following list: Intercalators that may affect the binding strength between the controller oligonucleotide and the first primer oligonucleotide, eg, MGB, naphthalene, etc. Like elements can also be used in the second region of the first primer.
  • Intercalators that may affect the binding strength between the controller oligonucleotide and the first primer oligonucleotide, eg, MGB, naphthalene, etc.
  • Like elements can also be used in the second region of the first primer.
  • the controller oligonucleotide may include in its first region non-nucleotide compounds, e.g. Linkers such as C3, C6, HEG linkers which link individual segments of the first region together.
  • non-nucleotide compounds e.g. Linkers such as C3, C6, HEG linkers which link individual segments of the first region together.
  • the controller oligonucleotide may bind by means of its second region to the first primer region of the first primer oligonucleotide, the binding being mediated essentially by hybridization of complementary bases.
  • the length of the second region of the controller oligonucleotide is matched to the length of the first region of the first primer oligonucleotide and is consistent with this in particular. It is between about 3-30 nucleotides, in particular between 5 and 20 nucleotides.
  • the sequence of the second region of controller oligonucleotide is in particular complementary to the first region of the first primer oligonucleotide. The degree of agreement in complementarity is between 80% and 100%, in particular between 95% and 100%, in particular at 100%.
  • the second portion of controller oligonucleotide specifically includes nucleotide modifications of one, which hinder the polymerase in extension of the first primer oligonucleotide, but do not block the formation of complementary double strands, or not substantially prevent, for example, 2 '-0-alkyl RNA analogs (for example, 2 '-0- Me, 2' -0- (2-methoxyethyl), 2 '-0-propyl, 2' -0-propargyl nucleotide modifications), LNA, PNA or morpholino nucleotide modifications.
  • individual nucleotide monomers are linked via 5 ' -3 ' linkage, but the alternative 5 ' -2 ' linkage between nucleotide monomers can also be used.
  • the sequence length and nature of the first and second regions of the controller oligonucleotide are particularly chosen such that the binding of these regions to the first primer oligonucleotide is reversible under reaction conditions, at least in one reaction step of the process. This means that although the controller oligonucleotide and the first primer oligonucleotide can specifically bind to one another, this bond should not lead to the formation of a complex of both elements that is permanently stable under reaction conditions.
  • an equilibrium between a bound complex form of controller oligonucleotide and the first primer oligonucleotide and a free form of individual components under reaction conditions should be sought or made possible at least in one reaction step. This ensures that at least a portion of the first primer oligonucleotides can be in free form under reaction conditions and can interact with the template to initiate a primer extension reaction. On the other hand, it will ensure that corresponding sequence regions of the controller oligonucleotide are available for binding with an extended primer oligonucleotide.
  • the proportion of free, single-stranded and thus reactive components can be influenced: by lowering the temperature, first primer oligonucleotides bind to the controller oligonucleotides, so that both participants bind a complementary double-stranded complex.
  • concentration of single-stranded forms of individual components can be lowered.
  • An increase in temperature can lead to the dissociation of both components into the single-stranded form.
  • the concentration of single-stranded forms in the reaction mixture can thus be influenced.
  • desired reaction conditions can be brought about during corresponding reaction steps. For example, by using temperature ranges approximately at the level of melting temperature of complexes of controller oligonucleotide / first primer oligonucleotide shares of each free forms of individual components can be influenced. In this case, the temperature used destabilizes complexes comprising controller oligonucleotide / first primer oligonucleotide, so that during this reaction step individual complex components are at least temporarily single-stranded and thus able to interact with other reactants.
  • a first sequence region of the controller oligonucleotide may be released from the double-stranded complex with a non-extended first primer, and thus may interact with the second sequence region of an extended first primer oligonucleotide, thereby initiating strand displacement.
  • release of a first, non-extended primer oligonucleotide from a complex comprising controller oligonucleotide / first primer oligonucleotide results in the first primer region becoming single-stranded and thus capable of interacting with the template such that primer extension by a polymerase can be initiated.
  • the temperature used does not have to correspond exactly to the melting temperature of the complex of controller oligonucleotide / first primer oligonucleotide. It is sufficient if the temperature is used in a reaction step, for example in the range of the melting temperature.
  • the temperature in one of the reaction steps comprises ranges of Tm +/- 10 ° C, in particular Tm +/- 5 ° C, in particular Tm +/- 3 ° C of the complex of controller oligonucleotide / first primer oligonucleotide.
  • Such a temperature can be set, for example, in the context of the reaction step, which comprises a sequence-specific strand displacement by the controller oligonucleotide.
  • reaction conditions are maintained over the entire duration of the amplification reaction in which there is equilibrium between a complex of controller oligonucleotide and the first Primer oligonucleotide and a free form of individual components is possible.
  • the ratio between a complex form of controller oligonucleotide and the first primer oligonucleotide and free forms of individual components can be influenced both by reaction conditions (eg temperature and Mg 2+ concentration) and by structures and concentrations of individual components.
  • the sequence length and nature of the first and second regions of the controller oligonucleotide are, in certain embodiments, chosen such that the ratio between a proportion of a free controller oligonucleotide is given under given reaction conditions (eg in the step of strand displacement by the controller oligonucleotide) and a portion of a controller oligonucleotide complexed with a first primer oligonucleotide comprises the following ranges: from 1: 100 to 100: 1, more preferably from 1:30 to 30: 1, more preferably from 1:10 to 10: 1.
  • the ratio between a proportion of a free first primer oligonucleotide and a proportion of a first primer oligonucleotide in complex with a controller oligonucleotide comprises ranges from 1: 100 to 100: 1, in particular from 1: 30 to 30: 1, in particular from 1 : 10 to 10: 1.
  • the concentration of the first primer oligonucleotide is higher than the concentration of the controller oligonucleotide.
  • the concentration of the first primer oligonucleotide is lower than the concentration of the controller oligonucleotide.
  • concentration of the controller oligonucleotide there is an excess of the controller oligonucleotide and the first primer oligonucleotide must be released for its effect of binding to the controller oligonucleotide by choosing an appropriate reaction temperature. In general, this is achieved by an increase in temperature, to sufficient concentrations of free forms of the first primer oligonucleotide.
  • the controller oligonucleotide may bind by means of its third region to at least one segment of the specifically synthesized extension product of the first primer oligonucleotide ( Figures 13 to 35). Binding is preferably by hybridization of complementary bases between the controller oligonucleotide and the extension product synthesized by the polymerase.
  • the sequence of the third region should in particular have a high complementarity to the extension product.
  • the sequence of the third region is 100% complementary to the extension product.
  • the binding of the third region takes place in particular on the segment of the extension product which directly adjoins the first region of the first primer oligonucleotide.
  • the segment of the extension product lies in the 5 ' segment of the entire extension product of the first primer oligonucleotide.
  • the binding of the third region of the controller oligonucleotide does not occur over the entire length of the extension product of the first primer oligonucleotide.
  • a segment remains unbound at the 3 ' end of the extension product. This 3 ' -terminal segment is necessary for the binding of the second primer oligonucleotide.
  • the length of the third region is adjusted to such an extent that the third region firstly binds to the 5 ' -terminal segment of the extension product, but on the other hand does not bind the 3 ' continuous segment of the extension product.
  • the total length of the third region of the controller oligonucleotide is from 2 to 100, in particular from 6 to 60, in particular from 10 to 40 nucleotides or their equivalents.
  • the controller oligonucleotide may enter into a complementary bond with the segment of the extension product over that length, thereby displacing this 5 ' segment of the extension product from binding with its complementary template strand.
  • the length of the 3 ' -terminal segment of the extension product which is not bound by the controller oligonucleotide comprises, for example, ranges between 5 and 100 nucleotides, in particular 5 and 60 nucleotides, in particular between 10 and 40, in particular between 15 and 30 nucleotides.
  • This 3 ' segment of the extension product is not displaced from the controller oligonucleotide from binding with the template strand. Even with the third region of the controller oligonucleotide fully bound to its complementary segment of the extension product, the first primer extension product may remain bound to the template strand via its 3 ' -terminal segment.
  • the bond strength of this complex is chosen in particular such that it can dissociate spontaneously under reaction conditions (step e), for example. This can be achieved, for example, by the melting temperature of this complex being selected from the 3 ' -terminal segment of the extension product of the first primer. Oligonucleotide and its template strand is approximately in the range of the reaction temperature or below the reaction temperature in a corresponding reaction step (reaction step e). With little stability of this complex in the 3 ' segment of the extension product, complete binding of the third region of the controller oligonucleotide to the 5 ' segment of the extension product results in rapid dissociation of the first primer extension product from its template strand.
  • the controller oligonucleotide as a whole has a suitable structure to perform its function: under appropriate reaction conditions, it is capable of sequence-specific displacement of the extended first primer oligonucleotide from binding with the template strand, thereby converting the template strand into single-stranded form, and thus for further binding with a novel first primer oligonucleotide and its target sequence-specific extension by the polymerase.
  • regions one, two and three of the controller oligonucleotide should be predominantly in single-stranded form under reaction conditions. Therefore, double-stranded self-complementary structures (e.g., hairpins) in these regions should be avoided as much as possible since they may degrade the functionality of the controller oligonucleotide.
  • double-stranded self-complementary structures e.g., hairpins
  • the controller oligonucleotide should not appear as a template in the method of the invention, therefore the first primer oligonucleotide, when attached to the controller oligonucleotide under reaction conditions, should not be extended by the polymerase.
  • the 3 ' end of the first primer oligonucleotide does not remain elongated when the first primer oligonucleotide binds to the controller oligonucleotide under reaction conditions.
  • the degree of blockage / inhibition / slowing / aggravation of the reaction may be between complete expression of this property (e.g., 100% blockage under given reaction conditions) and a partial expression of this property (e.g., 30-90% blockade under given reaction conditions).
  • the nucleotide modifications may include base modifications and / or sugar-phosphate-residue modifications.
  • the sugar-phosphate modifications are preferred because any complementary sequence of a controller oligonucleotide can be assembled by combining with conventional nucleobases.
  • the nucleotides with modifications in the sugar-phosphate residue, which can lead to hindrance or blockage of the synthesis of the polymerase include, for example: 2 '-0-alkyl modifications (eg, 2' -0-methyl, 2 '-0- (2- Methoxyethyl), 2 '-0-propyl, 2' -0-propargyl nucleotide modifications), 2 '-amino-2' -Deoxy- nucleotide modifications, 2 'amino-alkyl-2' -deoxy-nucleotide modifications , PNA, morpholino modifications, etc.
  • 2 '-0-alkyl modifications eg, 2' -0-methyl, 2 '-0- (2- Methoxyethyl), 2 '-0-propyl, 2' -0-propargyl nucleotide modifications
  • 2 '-amino-2' -Deoxy- nucleotide modifications e.g, 2 '-alkyl modifications
  • the blockade can be accomplished by either a single nucleotide modification or only by coupling multiple nucleotide modifications in series (e.g., as a sequence fragment consisting of modified nucleotides). For example, at least 2, in particular at least 5, in particular at least 10, of such nucleotide modifications can be coupled side by side in the controller oligonucleotide.
  • a controller oligonucleotide may comprise a uniform type of nucleotide modifications or may comprise at least two different types of nucleotide modification.
  • the location of such nucleotide modifications in the controller oligonucleotide is intended to prevent the polymerase from extending the 3 ' end of a first primer oligonucleotide bound to the controller oligonucleotide.
  • nucleotide modifications are located in the second region of the controller oligonucleotide. In certain embodiments, such nucleotide modifications are located in the third region of the controller oligonucleotide. In certain embodiments, such nucleotide modifications are located in the second and third regions of the controller oligonucleotide.
  • the second region of the controller oligonucleotide consists of at least 20% of its positions from such nucleotide modifications, in particular at least 50%.
  • the third region of the controller oligonucleotide consists of at least 20% of its positions from such nucleotide modifications, in particular at least 50%, in particular at least 90%.
  • the entire third region comprises nucleotide modifications that prevent a polymerase from extending a primer bound to such a region using the controller oligonucleotide as a template.
  • the entire third and second regions include such nucleotide modifications.
  • the entire first, second, and third regions include such nucleotide modifications.
  • the controller oligonucleotide may consist entirely of such nucleotide modifications.
  • Such modified controller oligonucleotides can be used, for example, in multiplex analyzes in which further primers are used. This is to prevent inadvertent primer extension reactions on one or more controller oligonucleotides.
  • sequence of nucleobases from these nucleotide modifications is adapted to the requirements of the sequence in each area.
  • the remaining portion can be made up of natural nucleotides, or nucleotide modifications which do not or only insignificantly prevent the polymerase function, for example DNA nucleotides, PTO nucleotides, LNA nucleotides, RNA nucleotides.
  • further modifications for example base modifications such as 2-amino-adenosine, 2-aminopurines, 5-methyl-cytosines, inosines, 5-nitroindoles, 7-deaza-adenosine, 7-deaza-guanosine, 5-propyl-cytosine, 5 Propyl uridine or non-nucleotide modifications such as dyes, or MGB modifications, etc.
  • the individual nucleotide monomers can be coupled to each other via conventional 5 ' -3 ' bond or else via 5 ' -2 ' bond.
  • a segment of the oligonucleotide controller nucleotide modification which prevents extension of the 3 ' end of a first primer oligonucleotide bound to the controller oligonucleotide by the polymerase is termed a "second blocking moiety".
  • the length of this segment may include between 1 to 50 nuclide modifications, especially between 4 and 30.
  • this segment may be located in the controller oligonucleotide such that the 3 ' end of the bound first primer oligonucleotide is in this segment.
  • this segment can span regions two and three.
  • no linker structures or spacer structures such as C3, C6, HEG linkers are used to prevent the extension of the 3 ' end of a first primer oligonucleotide bound to the controller oligonucleotide.
  • a controller oligonucleotide in its third region comprises at least one component of the detection system (eg, fluorescent reporter or fluorescent quencher or a donor fluorophore).
  • the position of this component is in certain embodiments at the 5 ' end of the controller oligonucleotide. In another embodiment, this component is in the inner sequence segment of the third region.
  • the distance up to the 5 ' end of the controller oligonucleotide may be between 2 to 50 nucleotides, in particular between 2 and 20, in particular between 2 and 10 nucleotides.
  • the controller oligonucleotide may comprise, in addition to regions one, two and three, further sequence segments which, for example, flank the abovementioned regions in the 5 ' segment or 3 ' segment of the controller oligonucleotide.
  • sequence elements can be used, for example, for other functions, such as, for example, interaction with probes, binding to solid phase, etc. In particular, such regions do not disturb the function of regions one to three.
  • the length of these flanking sequences may be, for example, between 1 to 50 nucleotides.
  • a controller oligonucleotide may comprise at least one element for immobilization on a solid phase, eg a biotin residue.
  • a controller oligonucleotide may include at least one element for detection, eg, a fluorine dye.
  • strand displacement of the template strands is affected by newly synthesized strands.
  • the strand displacement and / or separation is either slowed down quantitatively or completely eliminated. It is thus not at all or less often the transfer of primer binding sites in the single-stranded state. Thus, there are no or only few primer binding sites available for a new interaction with primers. Thus, the system of both primer extension products is rarely put into an active state or an active state is not reached.
  • the efficiency of the double-stranded opening of the newly synthesized primer extension products after each individual synthesis step affects the potentially achievable yields in subsequent cycles: the fewer free / single-stranded primer binding sites of a nucleic acid chain to be amplified at the beginning of a synthesis step The lower the number of newly synthesized strands of the nucleic acid chain to be amplified in this step. In other words, the yield of a synthesis cycle is proportional to the amount of primer binding sites available for interaction with corresponding complementary primers. Overall, this can be realized a control circuit.
  • This control circuit corresponds to a real-time / on-line control of synthesized fragments: Sequence control takes place in the reaction mixture while the amplification takes place. This sequence control follows a predetermined pattern and the oligonucleotide system (by the strand-opening action of the controller oligonucleotide) can distinguish between "correct” and "non-correct” states without external interference. In the correct state, the synthesis of sequences is continued; in the incorrect state, the synthesis is either slowed down or completely prevented. The resulting differences in yields of "correct” and “incorrect” sequences after each step affect the entire amplification comprising a variety of such steps. In exponential amplification, this dependence is exponential so that even slight differences in efficiency in a single synthesis cycle due to the sequence differences may signify a significant time delay in total amplification, or cause the complete absence of detectable amplification in a given time frame.
  • the displacement of the second primer extension product from binding with the first primer extension product by means of a sequence-dependent strand displacement by the controller oligonucleotide forms a partial step in the amplification.
  • the reaction conditions during this step are adjusted accordingly.
  • the reaction temperature and the reaction time are chosen so that the reaction can take place successfully.
  • strand displacement by the controller oligonucleotide proceeds until binding / dissociation of the second primer extension product from binding with the first primer extension product.
  • Such dissociation of the 3 ' segment of the first primer extension product from complementary portions of the second primer extension product may occur spontaneously as part of a temperature-dependent / temperature-related separation of both primer extension products.
  • Such dissociation has a favorable effect on the kinetics of the amplification reaction and can be influenced by the choice of reaction conditions, for example by means of temperature conditions. The temperature conditions are therefore chosen such that successful strand displacement by complementary binding of the controller oligonucleotide favors dissociation of the second primer extension product from the 3 ' segment of the first primer extension product.
  • This embodiment is not a "classical" PCR, but the thermally induced dissociation of the 3 'segment runs in temperature ranges well below the typical strand separation inducing reactions of> 90 ° C of classical PCR.
  • the strand displacement by the controller oligonucleotide proceeds until the detachment / dissociation of a 3 ' segment of the second primer extension product (P2.1 -Ext) from the complementary binding with the first primer extension product (P1 .1- Ext), wherein this 3 ' segment of the second primer extension product (P2.1 -Ext) comprises at least a complementary region to the first primer and a complementary segment to the first primer extension product (P1 .1-Ext), which is only in the enzymatic synthesis originated.
  • a new primer oligonucleotide can be linked to its primer binding site of this single stranded sequence segment of the complexed (P2.1 -ext) bind under reaction conditions and thus initiate a synthesis of a new first primer extension product (P1.2-Ext) by a polymerase.
  • this reaction proceeds at a reduced rate, since the 3 ' segment of the P2.1 -Ext is not permanently single-stranded, but is in competitive behavior with the controller oligonucleotide and thus alternately single-stranded and double-stranded states by binding to the P1 1 - Ext.
  • Such dissociation has a favorable effect on the kinetics of the amplification reaction and may be influenced by the choice of reaction conditions, e.g. by means of temperature conditions.
  • the involvement of the polymerase-mediated synthesis-dependent strand displacement in the dissociation of P1.1 -Ext and P2.1 -Ext has a favorable effect on strand separation.
  • the temperature in this step includes, for example, ranges from 15 ° C to 75 ° C, especially from 30 ° C to 70 ° C, especially from 50 ° C to 70 ° C.
  • the controller oligonucleotide may be attached to the first primer. Extend extension product to the 3 ' segment of the first primer extension product and displace the second primer extension product. The second primer extension product thus remains in association with the 3 ' segment of the first primer extension product.
  • This strength of this compound can be influenced by temperature. Upon reaching a critical temperature, this compound can decay and dissociate both primer extension products. The shorter the sequence of the 3 ' segment, the more unstable this compound and the lower the temperature which causes spontaneous dissociation.
  • a spontaneous Disretion can be achieved for example in the temperature range, which is approximately at the melting temperature.
  • the temperature of the strand displacement steps through the controller oligonucleotide is at about the melting temperature (Tm +/- 3 ° C) of the complex comprising the 3 ' segment of the first primer extension product that is not bound by controller oligonucleotide , and the second primer oligonucleotide and the second primer extension product, respectively.
  • the temperature of the strand displacement steps through the controller oligonucleotide is at about the melting temperature (Tm +/- 5 ° C) of the complex comprising the 3 ' segment of the first primer extension product that is not bound by the controller oligonucleotide , and the second primer oligonucleotide and the second primer extension product, respectively.
  • the temperature of the steps of strand displacement by the controller oligonucleotide is above the melting temperature of the complex comprising the 3 ' segment of the first primer extension product that is not bound by the controller oligonucleotide and the second primer oligonucleotide second primer extension product.
  • a temperature includes temperature ranges from about Tm + 5 ° C to Tm + 20 ° C, especially from Tm + 5 ° C to Tm + 10 ° C.
  • a first primer extension product comprises a 3 ' segment which is not bound by the controller oligonucleotide, and which
  • a spontaneous dissociation is usually already achieved at temperature ranges between 40 ° C and 65 ° C. Higher temperatures also lead to dissociation.
  • a first primer extension product comprises a 3 ' segment which is not bound by the controller oligonucleotide, and which
  • Sequence lengths from 15 to about 25 nucleotides.
  • a spontaneous dissociation usually already be achieved at temperature ranges between 50 ° C and 70 ° C. Higher temperatures also lead to dissociation.
  • a first primer extension product comprises a 3 ' segment that is not bound by the controller oligonucleotide and that has sequence lengths of from 20 to about 40 nucleotides.
  • a spontaneous dissociation usually already at temperature ranges between 50 ° C and 75 ° C can be achieved. Higher temperatures also lead to dissociation.
  • composition of the 3 ' segment of the first primer extension product and optionally an introduction of melting temperature-influencing oligonucleotide modifications (eg MGB) or reaction conditions (eg TPAC, betaine)) can influence the choice of temperature.
  • An appropriate adaptation can therefore be made.
  • all steps of the amplification proceed under stringent conditions that prevent or slow down the formation of nonspecific products / by-products.
  • stringent conditions include, for example, higher temperatures, for example above 50 ° C.
  • the single steps of strand displacement by controller oligonucleotides proceed at the same temperature as the synthesis of the first and second primer extension products.
  • the individual steps of strand displacement by controller oligonucleotides occur at temperature that differs from the temperature of the respective synthesis of the first and second primer extension products.
  • synthesis of the first primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature.
  • synthesis of the second primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature.
  • the concentration of the controller oligonucleotide comprises ranges from 0.01 pmol / l to 50 pmol / l, in particular from 0.1 pmol / l to 20 pmol / l, in particular from 0.1 pmol / l to 10 pmol / l.
  • An oligonucleotide capable of binding with its 3 ' segment to a substantially complementary sequence within the nucleic acid or its equivalents to be amplified and initiating a specific second primer extension reaction (Figs. 14, 27 to 29, 55, 68 to 71).
  • This second primer oligonucleotide is thus capable of binding to the 3 ' segment of a first specific primer extension product of the first primer oligonucleotide and initiating polymerase-dependent synthesis of a second primer extension product.
  • every second primer oligonucleotide is specific for each nucleic acid to be amplified.
  • the second primer oligonucleotide should be able to be copied in the reverse synthesis and also serves as a template for the synthesis of the first primer extension product.
  • the length of the second primer oligonucleotide can be between 15 and 100 nucleotides, in particular between 20 and 60 nucleotides, in particular between 30 and 50 nucleotides.
  • the nucleotide building blocks are in particular linked to each other via conventional 5'-3 'bond or Phosphonick- Phosphothioester bond.
  • Such a primer oligonucleotide can be chemically synthesized in the desired form.
  • the second primer oligonucleotide may include nucleotide monomers that do not or only slightly affect the function of the polymerase, including, for example:
  • Modified nucleotides 2-amino-dA, 2-thio-dT or other nucleotide modifications with aberrant base pairing (e.g., universal base pairs such as inosine 5-nitroindole).
  • aberrant base pairing e.g., universal base pairs such as inosine 5-nitroindole.
  • the 3 'OH end of this range in particular free of modifications and has a functional 3' -OH group, that can be recognized by the polymerase, and matrizenabphasenig extended.
  • the 3 ' segment of the second primer comprises at least one phosphorothioate compound such that no degradation from the 3 ' end of the primer by the 3 ' exonuclease activity of polymerases can occur.
  • the second primer oligonucleotide can be used in several partial steps. First and foremost, it performs a primer function in the amplification.
  • the primer extension reaction is carried out using the first primer extension product as a template.
  • the second primer oligonucleotide may use the starting nucleic acid chain as a template at the beginning of the amplification reaction.
  • the second primer oligonucleotide may be used in the preparation / provision of a starting nucleic acid chain.
  • the second primer serves as the initiator of the synthesis of the second primer extension product using the first primer extension product as template.
  • the 3 ' segment of the second primer comprises a sequence which can bind predominantly complementary to the first primer extension product.
  • Enzymatic extension of the second primer oligonucleotide using the first primer extension product as template results in the formation of the second primer extension product.
  • Such synthesis typically occurs in parallel with displacement of the controller oligonucleotide from its binding with the first primer extension product. This displacement is predominantly due to the polymerase and may be partially due to the second Primer oligonucleotide take place.
  • Such a second extension product comprises the target sequence or its segments.
  • the sequence of the copiable portion of the first primer oligonucleotide is recognized by the polymerase as a template and a corresponding complementary sequence is synthesized.
  • This sequence is located in the 3 ' segment of the second primer extension product and comprises the primer binding site for the first primer oligonucleotide.
  • the synthesis of the second primer extension product occurs until the stop position in the first primer oligonucleotide.
  • this product is bound to the first primer extension product to form a double-stranded complex.
  • the second primer extension product is sequence-specifically displaced from this complex by the controller oligonucleotide. Again, after successful strand displacement by the controller oligonucleotide, the second primer extension product can itself serve as a template for the synthesis of the first primer extension product.
  • the second primer oligonucleotide can serve as the initiator of the synthesis of the second primer extension product starting from the starting nucleic acid chain at the beginning of the amplification.
  • the sequence of the second primer is completely complementary to the corresponding sequence segment of a starting nucleic acid chain.
  • the sequence of the second primer oligonucleotide is only partially complementary to the corresponding sequence segment of a starting nucleic acid chain. However, this aberrant complementarity is not intended to prevent the second primer oligonucleotide from starting a predominantly sequence-specific primer extension reaction.
  • the respective differences in complementarity of the second primer oligonucleotide to the respective position in the starting nucleic acid chain are in particular in the 5 ' segment of the second primer oligonucleotide, so that in the 3 ' segment a predominantly complementary base pairing and initiation of the synthesis is possible.
  • the first 4-10 positions in the 3 ' segment should be fully complementary to the template (starting nucleic acid chain).
  • the remaining nucleotide positions may differ from a perfect complementarity.
  • the extent of perfect complementarity in the 5 ' segment can range between 10% to 100%, especially between 30% and 100% of the base composition.
  • this deviation encompasses a complete complementarity in the 5 ' segment of 1 to 40, in particular 1 to 20 nucleotide positions.
  • the second primer oligonucleotide binds only to its 3 ' segment to the starting nucleic acid chain, but not to its 5 ' segment.
  • the length of such a 3 ' - segment of the second primer oligonucleotide fully complementary to the starting nucleic acid chain comprises regions between 6 and 40 nucleotides, in particular between 6 and 30 nucleotides, in particular between 6 and 20.
  • the length of a corresponding, not complementary to the starting nucleic acid chain 5 ' segment of the second primer oligonucleotide comprises regions between 5 and 60, in particular between 10 and 40 Nucleotides.
  • the second primer oligonucleotide can thus initiate synthesis of a starting nucleic acid.
  • sequence sections of the second primer oligonucleotide are copied from the polymerase so that again in subsequent synthesis cycles, a fully complementary primer binding site is formed within the first primer extension product for binding of the second primer oligonucleotide and is available in subsequent synthesis cycles.
  • the second primer oligonucleotide may be used in the preparation of a starting nucleic acid chain.
  • a second primer oligonucleotide can bind to a nucleic acid (for example a single-stranded genomic DNA or RNA or its equivalents comprising a target sequence) predominantly / preferably sequence-specifically and initiate a template-dependent primer extension reaction in the presence of a polymerase.
  • the binding position is chosen such that the primer extension product comprises a desired target sequence.
  • the extension of the second primer oligonucleotide results in a strand which has a sequence complementary to the template.
  • Such a strand can be detached from the template (eg by heat or alkali) and thus converted into a single-stranded form.
  • a single-stranded nucleic acid chain can serve as a starting nucleic acid chain at the beginning of the amplification.
  • Such a start nucleic acid chain comprises in its 5 ' segment the sequence parts of the second primer oligonucleotide, furthermore it comprises a target sequence or its equivalents and a primer binding site for the first primer oligonucleotide. Further steps are explained in the section "Starting nucleic acid chain".
  • the second primer oligonucleotide comprises sequence portions which can bind complementarily and sequence specifically to a sequence segment of a target sequence and initiate / support a successful primer extension reaction by the polymerase.
  • the length of such a sequence segment comprises regions of 6 and 40 nucleotides, in particular of 8 to 30 nucleotides, in particular of 10 to 25 nucleotides.
  • the second primer oligonucleotide comprises copyable sequence segments in its 3 ' and 5 ' segments which are copied from the polymerase upon synthesis of the first primer extension product.
  • all sequence sections of the second primer are copied from the polymerase. This results in the formation of a primer binding site in the 3 ' segment of the first primer extension product.
  • the second primer oligonucleotide corresponds in length to the 3 ' segment of the first primer extension product that is not bound by the controller oligonucleotide.
  • the 3 ' end of such a second primer oligonucleotide is adjacent to the controller oligonucleotide which is adjacent to the first oligonucleotide Primer extension product is bound.
  • Extension of such a primer is done using the first primer extension product as a template. Upon extension of such a primer, the displacement of the controller oligonucleotide from binding with the first primer extension product occurs via polymerase-dependent strand displacement.
  • the second primer oligonucleotide with its copiable sequence portions is shorter than the 3 ' segment of the first primer extension product that is not bound by the controller oligonucleotide.
  • the complex comprising the second primer oligonucleotide and the first primer extension product, there is a single-stranded portion of the first primer extension product between the 3 ' end of such primer and the controller oligonucleotide bound to the first primer extension product.
  • Extension of such a primer is done using the first primer extension product as a template. Upon extension of such a primer, the displacement of the controller oligonucleotide from binding with the first primer extension product occurs via polymerase-dependent strand displacement.
  • the second primer oligonucleotide with its copatible portions is longer than the 3 ' segment of the first primer extension product that is not bound by the controller oligonucleotide.
  • the 3 ' segment of the second primer and the 5 ' segment of the controller oligonucleotide compete for binding to the first primer extension product.
  • the binding of the 3 ' segment of the second primer to the first primer extension product required for initiation of the synthesis takes place with simultaneous partial displacement of the 5 ' segment of the controller oligonucleotide.
  • the extension of such a primer is performed using the first primer extension product as a template.
  • the displacement of the controller oligonucleotide from binding with the first primer extension product occurs via polymerase-dependent strand displacement.
  • the sequence length of the 3 ' segment of the second primer oligonucleotide which displaces the 5 ' segment of the controller oligonucleotide may comprise the following ranges: 1 to 50 nucleotides, in particular 3 to 30 nucleotides, in particular 5 to 20 nucleotides.
  • second primer oligonucleotides of greater length which exceeds the length of the 3 ' segment of the first primer extension product, is advantageous in some embodiments.
  • Such embodiments include, for example, a first primer extension product having a 3 ' segment that is not bound by the controller oligonucleotide, having a length of 5 to 40 nucleotides, more preferably 10 to 30 nucleotides.
  • a longer second primer oligonucleotide provides improved sequence specificity upon initiation of the synthesis.
  • the binding strength of the second primer oligonucleotide to its primer binding site depends on the length of the primer. In general, longer second primer oligonucleotides can be used at higher reaction temperatures.
  • sequences of the first, second primer oligonucleotide and the controller oligonucleotide are matched to one another such that side reactions, e.g. Primer-dimer formation, minimized.
  • sequence of the first and second primer oligonucleotides are adapted to each other such that both primer oligonucleotides are incapable of undergoing an amplification reaction in the absence of an appropriate template and / or target sequence and / or starting sequence. Start nucleic acid chain.
  • the second primer oligonucleotide does not comprise a primer binding site for the first primer oligonucleotide and the first primer oligonucleotide does not comprise a primer binding site for the second primer oligonucleotide.
  • the primer sequences comprise extended self-complementary structures (self-complement).
  • the synthesis of the second primer extension product is a primer extension reaction and forms a substep in the first amplification.
  • the reaction conditions during this step are adjusted accordingly.
  • the reaction temperature and the reaction time are chosen so that the reaction can take place successfully.
  • the particular preferred temperature in this step depends on the polymerase used and the binding strength of the respective second primer oligonucleotide to its primer binding site and includes, for example, ranges from 15 ° C to 75 ° C, especially from 20 to 65 ° C, in particular from 25 ° C to 65 ° C.
  • the concentration of the second primer oligonucleotide comprises ranges from 0.01 pmol / l to 50 pmol / l, in particular from 0.1 pmol / l to 20 pmol / l, in particular from 0.1 pmol / l to 10 pmol / l.
  • all steps of the amplification proceed under stringent conditions that prevent or slow down the formation of nonspecific products / by-products.
  • stringent conditions include, for example, higher temperatures, for example above 50 ° C.
  • sequence-specific primer oligonucleotides are preferably used in each case for the amplification of corresponding respective target sequences.
  • the synthesis of the first and second primer extension products proceeds at the same temperature. In certain embodiments, the synthesis of the first and second primer extension products proceeds at different temperatures. In certain embodiments, synthesis of the second primer extension product and strand displacement by controller oligonucleotide proceeds at the same temperature. In certain embodiments, synthesis of the second primer extension product and strand displacement by controller oligonucleotide proceeds at different temperatures.
  • template-dependent polymerases are used which are capable of strand displacement.
  • a large fragment of the Bst polymerase or its modifications can be used.
  • Klenow fragment, Vent exo minus polymerase, Deepvent exo minus DNA polymerase, large fragment of Bsu DNA polymerase, large fragment of Bsm DNA polymerase can be used.
  • Vent exo minus polymerase, Deepvent exo minus DNA (from NEB) and PyroPhage polymerase from Lucigen are thermostable enzymes with strand displacement activity.
  • polymerases are used which exhibit no activity at room temperature, so-called hot-start polymerases or warm-start polymerases.
  • hot-start polymerases or warm-start polymerases.
  • An example of this is provided by Bst 2.0 Polymerase Warm Start (from NEB).
  • the third primer oligonucleotide is identical to the first primer oligonucleotide of the first amplification system.
  • the third primer oligonucleotide is not identical to the first primer oligonucleotide of the first amplification system.
  • the length of the third primer oligonucleotide may be between 15 and 100 nucleotides, in particular between 20 and 60 nucleotides, in particular between 30 and 50 nucleotides.
  • the CG content is for example between 20% and 80%, in particular between 30 and 79%.
  • the nucleotide building blocks are in particular linked to each other via conventional 5'-3 'bond or Phosphodie- Phosphothioester bond. Such a primer oligonucleotide can be chemically synthesized in the desired form.
  • the third primer oligonucleotide may include nucleotide monomers that do not or only insignificantly affect the function of the polymerase, including, for example:
  • Modified nucleotides nuclease-resistant phosphorothioate compounds (PTO) modifications, LNA modifications, 2-amino-dA, 2-thio-dT or other nucleotide modifications with different base pairing (eg universal base pairs, such as inosine 5 -Nitroindol).
  • the 3 'OH end of the third oligonucleotide primer in particular free of modifications and a functional 3' has -OH group of the Polymerase detected and can be extended depending on the template.
  • the 3 ' segment of the third primer comprises at least one phosphorothioate compound, so that no degradation of the 3 ' end of the primer by 3 ' exonuclease activity of polymerases can take place.
  • the 3 ' end of the third primer is blocked.
  • a 3 ' phosphate group or with a C3 linker The activation of the 3 ' segment of the third primer oligonucleotide in these embodiments takes place only in the second amplification, for example using 3 ' -5 ' -exonuclease activity of the second polymerase.
  • the third primer oligonucleotide does not comprise any sequence segments which are capable of complementary binding to the first region of the controller oligonucleotide.
  • the third primer oligonucleotide ( Figures 15-31) can be positioned in various arrangements relative to the first amplification fragment 1.1 (the first amplification product 1.1 comprising a target sequence).
  • the third and fourth primers In order for the second amplification reaction to start using a first amplification fragment 1 .1 as template (starting nucleic acid chain 2.1), the third and fourth primers must bind within sequences predetermined by the first amplification fragment and be extended by the polymerase become.
  • the third primer oligonucleotide can bind to a strand of the first amplification fragment 1 .1.
  • the third primer oligonucleotide binds to the second primer extension product and can thereby initiate primer extension using a suitable polymerase and reaction conditions.
  • This binding of the third primer oligonucleotide is in certain embodiments preferably in the 3 ' segment of the second primer extension product.
  • the third primer oligonucleotide preferably comprises a sequence segment in its 3 ' region which can bind complementarily or predominantly complementarily to the second primer extension product under used reaction conditions such that the polymerase is capable of facilitating the synthesis of the third primer extension product initiate.
  • this segment comprises in particular ranges between 6 and 30 nucleotides, in particular between 8 and 25 nucleotides, in particular between 10 and 20 nucleotides, in particular between 10 and 15 nucleotides.
  • this segment is fully complementary to the corresponding segment of the second primer extension product.
  • this segment comprises at least one mismatch to the primer extension product.
  • the position of this mismatch is no closer than -4 position with respect to the 3 ' end of the third primer, in particular no closer than -5, in particular no closer than -6, in particular no closer than position -8 relative to the 3 ' end of the third primer oligonucleotide.
  • the third primer oligonucleotide can also bind to the controller oligonucleotide with this 3 ' segment, such is Mismatch weakened the binding of this segment to the controller oligonucleotide.
  • this segment is functional at the reaction conditions used does not irreversibly bind to the controller oligonucleotide and thus inactivate it.
  • the third primer oligonucleotide may bind predominantly complementary to the same sequence segment as the first primer oligonucleotide.
  • the position of the 3 ' end of the third primer oligonucleotide matches the position of the 3 ' end of the first primer oligonucleotide.
  • the third primer oligonucleotide may at least partially (with its 3 ' segment) bind complementary to a portion of the target sequence.
  • the 3 ' end of the third primer is thus within the second blocking unit of the controller oligonucleotide and can not be extended through the polymerase using the controller as a template ( Figure 20).
  • the third primer oligonucleotide binds predominantly complementary to the second primer extension product with its 3 ' end shifted relative to the 3 ' end of the first primer oligonucleotide.
  • the 3 ' end of the third primer oligonucleotide is shifted by at least one nucleotide in the 3 ' direction of the second primer extension product.
  • the segment of the second primer extension product to which the third primer oligonucleotide is capable of predominantly complementary binding is shifted in the 3 ' direction ( Figure 19).
  • the 3 ' end of the third primer oligonucleotide is shifted by at least one nucleotide in the 5 ' direction of the second primer extension product.
  • the segment of the second primer extension product, to which the third primer oligonucleotide can bind predominantly complementarily is shifted in the 5 ' direction (FIG. 21).
  • the binding position of the third primer oligonucleotide is shifted in the 5 ' direction of the second primer extension product so that the third primer oligonucleotide does not overlap the binding position of the first primer oligonucleotide of the first amplification system.
  • the segment of the second primer extension product to which the third primer oligonucleotide is capable of predominantly complementary binding is shifted in the 5 ' direction relative to the binding site of the first primer oligonucleotide ( Figure 22).
  • the third primer oligonucleotide thus comprises sequence segments which can bind complementarily to the controller oligonucleotide.
  • the segment of the controller oligonucleotide which is capable of complementary binding to the third primer oligonucleotide is referred to as the fourth region of the controller oligonucleotide.
  • the fourth blocking unit Figures 19-22. Their position depends on the potential binding position of the third primer oligonucleotide within the controller.
  • the third primer oligonucleotide is said to be capable of being copied upon reverse synthesis and also serves as a template for the synthesis of the fourth primer extension product.
  • the third primer oligonucleotide comprises, in certain embodiments, at least one sequence segment in its 3 ' region which is capable of predominantly complementary binding to the first target sequence. This segment can bind predominantly complementarily to the second primer extension product (comprising corresponding segments of the target sequence), which polymerase can perform a primer extension reaction.
  • the third primer oligonucleotide in certain embodiments, comprises at least one sequence segment in its 5 ' region which is not complementary to the target sequence.
  • This segment may, for example, comprise from 1 to 60 nucleotides and may serve, for example, for other purposes, for example barcoding, cloning, immobilization, probe binding, etc.
  • the sequence composition of this 5 ' region is adapted so as not to interfere with the second amplification.
  • the third primer oligonucleotide may comprise further modifications, eg fluorescent dyes (eg FAM, Cy5 etc.), fluorescence quenchers (eg BHQ1, BHQ2 etc), affinity tags (eg biotin, digoxigenin etc.).
  • the third primer oligonucleotide may comprise a linker (eg, C3 or HEG linker) such that its 5 ' region remains uncoupled from the polymerase.
  • the third primer is immobilized on a solid phase prior to the second amplification reaction.
  • the primer extension of this third primer oligonucleotide thus leads to the immobilization of the entire third primer extension product.
  • the concentration of the third primer oligonucleotide used may comprise, for example, ranges between 0.01 pmol / l to about 10 pmol / l, in particular between 0.1 pmol / l and about 2 pmol / l.
  • the fourth primer oligonucleotide is identical to the second primer oligonucleotide of the first amplification system.
  • the fourth primer oligonucleotide is not identical to the second primer oligonucleotide of the first amplification system.
  • the length of the fourth primer oligonucleotide can be between 15 and 100 nucleotides, in particular between 20 and 60 nucleotides, in particular between 30 and 50 nucleotides.
  • the CG content is for example between 20% and 80%, in particular between 30 and 79%.
  • the nucleotide building blocks are in particular linked to each other via conventional 5'-3 'phosphodiester bond or Phosphothioester bond. Such a primer oligonucleotide can be chemically synthesized in the desired form.
  • the fourth primer oligonucleotide may include nucleotide monomers that do not or only insignificantly affect the function of the polymerase, including, for example:
  • Modified nucleotides nuclease-resistant phosphorothioate compounds (PTO) modifications, LNA modifications, 2-amino-dA, 2-thio-dT or other nucleotide modifications with different base pairing (eg universal base pairs, such as inosine 5 -Nitroindol).
  • the 3 'OH end of the fourth oligonucleotide primer in particular free of modifications and has a functional 3' -OH group, that can be recognized by the polymerase, and template-extended.
  • the 3 ' segment of the fourth primer comprises at least one phosphorothioate compound such that no degradation from the 3 ' end of the primer by the 3 ' exonuclease activity of polymerases can occur.
  • the 3 ' end of the fourth primer is blocked.
  • a 3 ' phosphate group or with a C3 linker The activation of the 3 ' segment of the fourth primer oligonucleotide in these embodiments takes place only in the second amplification, for example using 3 ' -5 ' -exonuclease activity of the second polymerase.
  • the fourth primer oligonucleotide does not comprise any sequence segments which are capable of complementary binding to the first region of the controller oligonucleotide.
  • the fourth primer oligonucleotide ( Figures 15-31) can be positioned in various arrangements relative to the first amplification fragment 1.1 (the first amplification product 1.1 comprising a target sequence).
  • the third and fourth primers In order for the second amplification reaction to start using a first amplification fragment 1 .1 as template (starting nucleic acid chain 2.1), the third and fourth primers must bind within sequences predetermined by the first amplification fragment and be extended by the polymerase become.
  • the fourth primer oligonucleotide can bind to a strand of the first amplification fragment 1.1.
  • the fourth primer oligonucleotide binds to the first primer extension product and can thereby initiate primer extension using a suitable polymerase and reaction conditions.
  • This binding of the fourth primer oligonucleotide is in certain embodiments preferably in the 3 ' segment of the first primer extension product.
  • the fourth primer oligonucleotide preferably comprises a sequence segment in its 3 ' region which can bind complementary or predominantly complementary to the first primer extension product under reaction conditions used so that the polymerase is capable of facilitating the synthesis of the fourth primer extension product to initiate.
  • the length of this segment comprises in particular ranges between 6 and 40 nucleotides, in particular between 8 and 30 nucleotides, in particular between 10 and 25 nucleotides, in particular between 10 and 20 nucleotides.
  • this segment is fully complementary to the corresponding segment of the first primer extension product.
  • this segment comprises at least one mismatch to the first primer extension product.
  • the position of this mismatch is no closer than -4 position with respect to the 3 ' end of the fourth primer, in particular not closer than -5, in particular not closer than -6, in particular not closer than position -8 relative to the 3 ' . End of the fourth primer oligonucleotide.
  • the position of the binding of this segment to the first primer extension product may be chosen differently (Figs. 23-31, 57).
  • the fourth primer oligonucleotide may bind predominantly complementary to the same sequence segment as the second primer oligonucleotide.
  • the position of the 3 ' end of the fourth primer oligonucleotide matches the position of the 3 ' end of the second primer oligonucleotide.
  • the fourth primer oligonucleotide comprises at least in part (with its 3 ' segment) a portion of the target sequence.
  • the fourth primer oligonucleotide binds predominantly complementary to the first primer extension product, with its 3 ' end being shifted with respect to the 3 ' end of the second primer oligonucleotide.
  • the 3 ' end of the fourth primer oligonucleotide is shifted by at least one nucleotide in the 3 ' direction of the first primer extension product.
  • the segment of the first primer extension product to which the fourth primer oligonucleotide is capable of predominantly complementary binding is displaced in the 3 ' direction ( Figure 1).
  • the 3 ' end of the fourth primer oligonucleotide is shifted by at least one nucleotide in the 5 ' direction of the first primer extension product.
  • the segment of the first primer extension product to which the fourth primer oligonucleotide is capable of predominantly complementary binding is shifted in the 5 ' direction.
  • the fourth primer oligonucleotide does not comprise any sequence segments which can bind complementarily to the controller oligonucleotide.
  • the fourth primer oligonucleotide should be able to be copied in reverse synthesis and also serves as a template for in the context of the synthesis of the third primer extension product.
  • the fourth primer oligonucleotide in certain embodiments, comprises at least one sequence segment in its 3 ' region which comprises portions of the first target sequence and thus can bind to a strand which is complementary to the target sequence.
  • This segment can be predominantly complementary to the first primer extension product (comprising corresponding complementary segments to the target sequence), which polymerase can perform a primer extension reaction.
  • the fourth primer oligonucleotide in certain embodiments, comprises at least one sequence segment in its 5 ' region which does not comprise sequence segments of the first target sequence.
  • This segment may, for example, comprise from 1 to 60 nucleotides and may for example serve other purposes, for example "barcoding", cloning, immobilization, probe binding, etc.
  • the sequence composition of this 5 ' region is adapted so as not to interfere with the second amplification ,
  • the fourth primer oligonucleotide may comprise further modifications, eg fluorescent dyes (eg FAM, Cy5 etc.), fluorescence quenchers (eg BHQ1, BHQ2 etc), affinity tags (eg biotin, digoxigenin etc.)
  • fluorescent dyes eg FAM, Cy5 etc.
  • fluorescence quenchers eg BHQ1, BHQ2 etc
  • affinity tags eg biotin, digoxigenin etc.
  • Primer oligonucleotide include a linker (eg C3 or HEG linker) so that its 5 ' region remains uncoupled from the polymerase.
  • the fourth primer is immobilized on a solid phase prior to the second amplification reaction.
  • the primer extension of such a fourth primer oligonucleotide thus leads to immobilization of the entire fourth primer extension product.
  • the concentration of the fourth primer oligonucleotide used may comprise, for example, ranges between 0.01 pmol / l to about 10 pmol / l, in particular between 0.1 pmol / l and about 2 pmol / l.
  • thermostable template-dependent DNA polymerases are used, which have a 5'-3 'exonuclease activity.
  • Taq polymerase and / or its formulations and / or their modifications are used to carry out the second amplification.
  • thermostable template-dependent DNA polymerases having strand-displacement activity are used.
  • Vent Exo minus from NEB
  • PyroPhage Polymerase from Lucigene
  • SD Polymerase from Bioron.
  • thermostable template-dependent DNA polymerases are used which have a 3 ' -5 ' proof reading activity.
  • Vent exo plus Deep Vent exo plus (from NEB), Pfu Polymerase (Jena Biosciences), Phusion Polymerase (NEB).
  • thermostable template-dependent DNA polymerases are used which are conjugated to another protein, for example to increase the polymerase's processivity.
  • Phusion Polymerase NEB
  • Primer oligonucleotides comprising additional sequence segments:
  • first primer and the second primer can be considered as so-called “base structure of the primer” or “minimal structure of the primer”.
  • Such basic structures of oligonucleotides with primer function comprise sequence segments which are essential for the execution of the amplification reaction.
  • Method are advantageous, for example, the first and second region of the first primer.
  • Such a basic structure of the primer can be extended by additional, additional sequence segments.
  • additional sequence segments include structures which, while not necessary for the performance of the amplification process, may nevertheless be useful for other tasks.
  • Such additional sequence segments may optionally be introduced into a primer and used for further functions or reactions. This allows the polymerase synthesized primer extension products (starting from, for example, the first and / or the second primer) to be linked to such sequences segments. This achieves integration of such additional sequence segments and primer extension products into a molecular structure. Such integration may be advantageous in certain embodiments.
  • a variety of applications for primer sequences with additional sequence segments are known to one skilled in the art.

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Abstract

La présente invention concerne un procédé pour l'amplification d'un acide nucléique avec une spécificité améliorée par l'utilisation d'oligonucléotides d'amorce et de nucléotides régulateurs particuliers, dans une première amplification les oligonucléotides régulateurs permettant d'une manière spécifique de séquence une ouverture de brin dans le produit d'amplification. L'invention concerne an outre un kit correspondant pour l'exécution du procédé selon l'invention.
EP19712690.7A 2018-02-26 2019-02-26 Procédé pour l'amplification d'un acide nucléique avec spécificité améliorée Withdrawn EP3759248A1 (fr)

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EP18158722 2018-02-26
EP18159337 2018-02-28
DE102018001586.7A DE102018001586A1 (de) 2018-02-28 2018-02-28 Verfahren zur Detektion der Amplifikation einer Nukleinsäure mit verbesserter Spezifität
EP18195312.6A EP3530754A1 (fr) 2018-02-26 2018-09-18 Procédé d'amplification d'un acide nucléique à spécificité améliorée
PCT/EP2019/054767 WO2019162530A1 (fr) 2018-02-26 2019-02-26 Procédé pour l'amplification d'un acide nucléique avec spécificité améliorée

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US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US20040091923A1 (en) * 1993-07-23 2004-05-13 Bio-Rad Laboratories, Inc. Linked linear amplification of nucleic acids
US5882857A (en) * 1995-06-07 1999-03-16 Behringwerke Ag Internal positive controls for nucleic acid amplification
WO2002018616A1 (fr) * 2000-09-01 2002-03-07 Hitachi Chemical Co., Ltd. Ajustement du rendement d'amplification d'une matrice d'acide nucleique par pcr attenuee a l'aide d'oligonucleotides matrices-genocopies
CN101889096B (zh) * 2007-10-04 2015-10-21 联邦科学及工业研究组织 核酸扩增
CN104593483B (zh) * 2009-08-25 2018-04-20 伊鲁米那股份有限公司 选择和扩增多核苷酸的方法
EP3144396B1 (fr) * 2010-10-27 2020-01-01 President and Fellows of Harvard College Procédés d'utilisation d'amorces à séquence d'ancrage toehold en épingle à cheveux
US9115394B2 (en) * 2011-12-22 2015-08-25 Roche Molecular Systems, Inc. Methods and reagents for reducing non-specific amplification
DK3234188T3 (da) * 2014-12-15 2020-01-20 Cepheid Nukleinsyreamplificering med eksponentiel base, der er større end 2

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