WO2014122422A2 - Technique d'amplification - Google Patents

Technique d'amplification Download PDF

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Publication number
WO2014122422A2
WO2014122422A2 PCT/GB2014/050102 GB2014050102W WO2014122422A2 WO 2014122422 A2 WO2014122422 A2 WO 2014122422A2 GB 2014050102 W GB2014050102 W GB 2014050102W WO 2014122422 A2 WO2014122422 A2 WO 2014122422A2
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nucleotide
primer
nucleotides
amplification
concentration
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PCT/GB2014/050102
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WO2014122422A3 (fr
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Robert Powell
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Primerdesign Ltd
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Priority claimed from GBGB1302112.6A external-priority patent/GB201302112D0/en
Priority claimed from GB201308291A external-priority patent/GB201308291D0/en
Application filed by Primerdesign Ltd filed Critical Primerdesign Ltd
Publication of WO2014122422A2 publication Critical patent/WO2014122422A2/fr
Publication of WO2014122422A3 publication Critical patent/WO2014122422A3/fr

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    • 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
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the invention relates to nucleic acid amplification reactions and in particular to amplification reactions which amplify a specific sequence including a polymorphism such as allele specific nucleic acid amplifications, including in particular the polymerase chain reaction (PCR).
  • Allele specific PCR is a method for selectively amplifying DNA target sequences from a mixture of potential DNA target sequences that are very similar or that differ by only one nucleotide base. Such methods can be used to detect the presence of mutation using a wide range of reporting chemistries that are well known in amplification techniques such as PCR. The method can also be used to quantify the presence of mutations using real time PCR with a wide range of detection chemistries. Novel compositions for use in such reactions form a further aspect of the invention.
  • Nucleic acid amplification reactions such as PCR provide an invaluable tool for producing, detecting and analysing nucleic acids for a wide range of purposes.
  • the specificity of these types of reaction allows the detection of particular target sequences within a background of multiple different sequences.
  • the basic principle behind many nucleic acid amplification reactions is the use of short 'primer' sequences that are designed to specifically bind to a target sequence and are then extended by the enzymatic addition of individual nucleotides, which are present in excess in the reaction mixture to ensure efficient
  • mis-priming events there can sometimes be problems arising for example as a result of mis- priming events, where a primer sequence used in the amplification binds albeit imperfectly to the non-target sequence during the process. This non-target sequence is also amplified during the process giving rise to artefacts in the result.
  • the risk of mis-priming events is high where the sample contains sequences which are very similar to the target sequence. This increases the risk that a primer will bind a non-target sequence during the amplification reaction.
  • Such conditions apply in a wide range of fields including for instance, genetic analysis, pathogen detection, forensics, speciation for analysis or quality control purposes (for instance meat speciation) and the detection of genetically modified organisms.
  • the target sequence may differ from other sequences in the sample by only a small number of nucleotides or even a single nucleotide or single nucleotide polymorphism (SNP).
  • the ability to detect genetic mutations can be critical in the diagnosis and aetiology of a wide range of illness.
  • mutations can arise that are not present in the germ line.
  • the presence of these mutations can be critical to understanding the staging and possible drug resistance phenotype of the tumour.
  • these mutations may be present in only a small proportion of the tumour.
  • the biological samples from patients may contain healthy germ line tissue as well as tumour cells, it is apparent that the proportion of cells contain the mutation of interest can be very low and in the region of 1 in 1000 of even lower in some scenarios.
  • Allele specific PCR methods have a PCR primer target that sites the 3 'end of the PCR primer adjacent to the variant base.
  • the allele specific primer may be homologue to the either the wild type or the variant sequence and will therefore selectively amplify the cognate sequence.
  • a significant problem with allele specific PCR is that a single base change, even when it is placed at or near the 3 'end of the primer does not always confer complete specificity and mispriming can occur on the alternative allele.
  • the PCR reaction therefore often results in a mixture of amplified products.
  • Substitute nucleotides have been included in nucleic acid amplification reactions previously for decontamination purposes (USP5, 536,649 and USP6,248,522).
  • WO2005/076925 disclosures the substitution of a portion of a conventional nucleotide such as dTTP with an unconventional or substitute nucleotide such as dUTP in order to enhance specificity and sensitivity of a process by reduction of primer-aggregate formation.
  • Primer- dimer formation is a unilateral problem that can occur in any amplification reaction. There is no suggestion that such an approach would be particularly beneficial in amplification reactions such as allele specific amplification reactions where mis-priming is understood to be a major factor in disrupting the level of specificity achievable.
  • modified nucleotide analogues have been added to amplification reactions previously for a variety of purposes including generating a 'hot-start' mixture, where the analogue includes a thermolabile 3' group (WO2009/151921), for labelling the product by incorporation of a fluorescent or biotin label (see for example W099/42611 or Paul et al. BioTechniques,2010. 48:333-334).
  • WO02/21098 describes a process in which modified nucleotides are incorporated into an amplification product in order to generate cleavage sites, so that the product may be characterised following cleavage at the sites of the modified nucleotides.
  • modified nucleotides can be incorporated into amplification products during processes such as the PCR, it has also been recognised that they may reduce the efficiency of the incorporation since they are generally larger than the native bases and may not be easily accommodated by the polymerase enzymes.
  • WO2004/003228 describes a method of genotyping in which single or small number of nucleotides or modified nucleotides, in particular those able to act as extension terminators such as dideoxynucleotides or acylonucleotides, are used to extend a primer in a detectable manner, for genotyping purposes, but in this case, it is taught that the 3 'end of the primer must itself be modified, for example by introducing sulphur substitutions in the phosphate structure.
  • 'alkyl' refers to a saturated hydrocarbon chain that may be straight or branched. They suitably contain up to 20 carbon atoms for example from 1-15 carbon atoms, in particular from 1-10 carbon atoms.
  • 'alkenyl' and 'alkynyl' refer to an unsaturated straight or branched hydrocarbon chain that include at least one double or triple bond respectively. They suitably contain up to 20 carbon atoms for example from 2-15 carbon atoms, in particular from 2-10 carbon atoms.
  • Allele specific primer A PCR primer having the 3' terminal base at the same position as a single nucleotide polymorphism.
  • 'aryl' refers to aromatic cyclic hetercarbyl groups having for instance from 6-20 carbon atoms. They may be monocyclic or comprise two or more fused rings, for example a system comprising a fused 5- and 6- membered rings.
  • heteroaryl' refers to aryl groups that contain one or more heteroatoms.
  • “Enhance the specificity” means that the production of non-target sequences is reduced as compared to a similar reaction carried out in the presence of a conventional mixture comprising similar amounts of each of the relevant nucleotides (dATP, dCTP, dGTP and dTTP in the case of DNA amplification and dUTP in the case of RNA amplification).
  • 'functional group' refers to a reactive chemical moiety that may comprise or contain a heteroatom.
  • examples include halo (such as, fluoro, chloro or iodo), hydroxyl, alkoxy, nitro, cyano, thiol,thioalkyl, sulphone, sulphate, sulphide, phosphine, phosphate, haloalkyl such as trifluoromethyl, alkylsulphonyl, alkysulphinyl, carboxyl or salts or esters thereof (in particular alkyl esters), alkoxycarbonyl groups, oxo groups,
  • 'heteroatom' includes oxygen, sulphur, nitrogen and phosphorus.
  • hydrocarbyl' refers to organic chemical groups containing hydrogen and carbon atoms.
  • the allele specific primers that are structurally distinct from the intended allele specific primer that are present to prevent the mis-priming and amplification of the wrong allele by the intended allele specific primer.
  • modified nucleotide analogue refers to a moiety having a structure such that can bind to a nucleic acid in place of one of the naturally occurring bases cytosine, guanine, adenine, thymine or uracil, but is modified in its chemical structure as compared to those bases. Particular examples of the sorts of modifications that are envisaged are discussed below.
  • substitute nucleotide refers to a natural nucleotide which does not occur in the type of nucleic acid under consideration.
  • deoxy-uridine triphosphate(dUTP) is a 'substitute nucleotide' in an amplification of a DNA sequence
  • deoxy-thymidine triphosphate (dTTP) is the substitute nucleotide in an amplification of an RNA sequence.
  • Touchdown PCR A PCR thermocycling method that starts with a high annealing temperature and drops the annealing temperature incrementally with each cycle until the cycling Tm of the primer is reached.
  • cDNA Complementary DNA gDNA. Genomic DNA from a biological source
  • nucleic acid amplification reaction aimed at detecting sequences that contain polymorphisms or alleles can be enhanced by changing the composition of the nucleic acids, in particular by adding modified nucleotides to the mixture and/or by controlling and in particular restricting the amount of nucleotides available in the reaction.
  • the invention provides a method for carrying out a nucleic acid amplification reaction to amplify a specific sequence which includes a polymorphism, which method comprises carrying out a nucleic acid amplification reaction using a nucleic acid amplification reaction mixture which comprises a nucleic acid primer having a 3 'end that binds at or near the site of the polymorphism, and a set of nucleotides able to effect amplification of the target sequence and wherein the composition of the nucleotides is selected so as to enhance the specificity of the primer for the specific sequence.
  • the invention provides a method for carrying out a nucleic acid amplification reaction to amplify a specific sequence which includes a polymorphism, which method comprises carrying out a nucleic acid amplification reaction wherein a nucleic acid primer having a 3 'end that binds at or near the site of the polymorphism is employed, and wherein the amount of at least one nucleotide present in the reaction mixture is selected so that the specificity of the amplification reaction is increased.
  • the composition of nucleotides present in the reaction mixture used in the amplification is selected so as to enhance the specificity of the primer.
  • the nucleic acid amplification reaction may be any reaction in which a primer is extended by enzymatic addition of one or more nucleotides to it whilst that primer is bound or hybridised to a target sequence.
  • Such reactions include reactions that utilise thermal cycling such as the polymerase chain reaction (PCR) and ligase chain reaction (LCR) as well as isothermal amplification reactions such as nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), transcription mediated amplification (TMA), Loop- Mediated Isothermal Amplification (LAMP) and rolling circle amplification, 3SR, ramification amplification (as described by Zhang et al., Molecular Diagnosis (2001) 6 No 2, p 141-150), recombinase polymerase amplification (available from TwistDx) and others.
  • the nucleic acid amplification is a PCR.
  • the allele specific nucleic acid amplification reaction primer used in the amplification reaction is designed to fully hybridise to the target sequence so that the 3 ' end of the primer is at or near the site of a target polymorphism in a sequence.
  • the nucleotide at the 3 'end of the primer directly overlies or binds to the polymorphic nucleotide in the target. This arrangement will maximize the chances that primer extension will only occur if there is a correct match between the nucleotide at the 3 'end of the primer and the corresponding base in the target sequence.
  • the primer will only extend if there is a 'match' between the nucleotide at the 3' end of the primer and the nucleotide at the site of the polymorphism in the target sequence, but this is not always the case, and mis-priming events can lead to artefacts and inaccurate results.
  • the primer will be designed to match either the native sequence or the sequence that contains the polymorphism and suitably the latter. In some instances, primers of both sequences may be present in the mixture but in such cases, the different primers will carry different labels, for instance different fluorescent labels to indicate which specific sequence has been amplified in any particular case.
  • the adjustment of the composition of the mixture of nucleotides in accordance with the invention may be effected in one of two basic ways (i) by adding to the mixture a substitute nucleotide or a modified nucleotide analogue and/or (ii) by reducing the concentration of at least one of the nucleotides present in the mixture below that which is regarded as optimum for the process.
  • the composition of the mixture of nucleotides is adjusted by adding to the mixture a substitute nucleotide or a modified nucleotide analogue in an amount sufficient to enhance the specificity of the amplification reaction. This is achieved by increasing the specificity of the primer for the specific sequence such as the correct genotype during the amplification.
  • the amplification reaction mixture will typically contain each of the conventional nucleotides (dNTPs) including dATP, dCTP, dGTP and dTTP when the amplification product is a DNA molecule, or dUTP where the amplification product is an RNA molecule, but will additionally comprise a substitute nucleotide or a modified nucleotide analogue.
  • dNTPs conventional nucleotides
  • the substitute nucleotide or modified nucleotide analogue is one which competes for binding with a nucleotide that is incorporated in the region of the 3 'end of the primer, for instance within 1-10 for example 1-5 bases of the 3' end, and in particular competes with a nucleotide which binds at the 3 'end of the primer during the amplification.
  • the concentration of at least one of the nucleotides that is incorporated in the region of the 3 'end of the primer for instance within 1-10 for example 1-5 bases of the 3' end, and in particular is the nucleotide which binds at the 3 'end of the primer during the amplification reaction is reduced.
  • the modified nucleotide analogue will be the complement of the base directly adjacent the polymorphism in the target sequence in the 5 'direction.
  • Suitable modified nucleotide analogues for use in the invention are those which carry substituents at any available positions on the purine or pyrimidine ring of the base, such as a purine nucleotide with a chemical modification to the carbon atom at position 8, a 7- deazapurine nucleotide with a chemical modification to the carbon atom at position 7 or any pyrimidine nucleotide with a chemical modification attached to the carbon at position 5.
  • the base cytosine or uracil nucleotides may be substituted at the 5- and/or 6- positions on the pyrimidine ring, a thymine nucleotide may be chemically modified at the 6- position on the pyrimidine ring, an adenosine nucleotide may be substituted at the 2- and/or 8- positions on the purine ring and a guanine nucleotide may carry be substituted at the 8- position on the purine ring.
  • modified nucleotide analogues for use in the invention include those modified at the hydrogen-bonding positions of the purine or pyrimidine base, including base analogues such as 2-thiouracil and 2-aminopurine as shown below.
  • Such bases may also carry substituents at the available positions.
  • modified nucleotide analogues include 2-aminopurine-2'-deoxyriboxe-Triphosphate (2- aminopurine-drTP) and 2-Thio-2'deoxycytidine-5 '-triphosphate (2-Thio-dCTP).
  • Suitable substituents are functional groups or optionally substituted hydrocarbyl groups that may have heteroatoms interposed therein.
  • Suitable optional substituents for the hydrocarbyl groups are functional groups as defined above.
  • the hydrocarbyl groups suitably contain up to 20 carbon atoms, for instance up to 15 carbon atoms, or more suitably up to 10 carbon atoms. They are suitably arranged as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or cycloalkynyl groups as well as aryl groups or heteroaryl groups.
  • the modified nucleotide analogue carries an unsubstituted hydrocarbyl substituent and in particular a C 2 -io alkynyl group such as Octa-l,7-diynyl.
  • modified nucleotide analogues will be the triphosphate of any of the nucleosi
  • R 1 is a substituent as defined above, and in particular is a hydrocarbyl group such as an alkyl, alkenyl or alkynyl group having from 1 to 15 and suitably from 1 to 10 carbon atoms such as octa-l,7-diynyl.
  • R 1 is other than methyl.
  • Particular examples include 5-(Octa-l,7-diynyl)-5 -O- triphosphate-2 ' -deoxycytidine (C8-alkyne-dCTP) of formula (v).
  • R 1 may be a functional group such as a halo group and in particular iodo.
  • R 1 may be a functional group such as a halo group and in particular iodo.
  • R 1 may be a functional group such as a halo group and in particular iodo.
  • R 1 may be a functional group such as a halo group and in particular iodo.
  • R 1 may be the triphosphate derivative of compound of formula (i) where R 1 is iodo. This is 5 -iodo-2'-deoxyridine-5' -triphosphate or 5-iodo-dUTP of formula (v):
  • Particular examples include 5-(Octa-l,7-diynyl)-5 -0-triphosphate-2' -deoxycytidine (C8-alkyne-dCTP).
  • a substitute nucleotide in the amplification of DNA will be derived from the nucleoside dU of structure (vi), and in particular will be the triphosphate derivative dUTP.
  • a substitute nucleotide or a modified nucleotide analogue that appears effectively to slow down the primer extension process, in particular in the initial stages of that extension process, in accordance with the method of the invention, the risk of mis-priming events are reduced.
  • a substitute nucleotide or a modified nucleotide analogues are added to the reaction mix. They are added at a sufficient
  • reaction mix will otherwise contain the elements necessary for carrying out the amplification as would be understood in the art. They may comprise for example PCR 'mastermixes' that are available commercially.
  • the modified nucleotide analogue will be added in an amount which is at least 50% of that of each of the usual nucleotides, for instance from 0.6 to 10 times the amount of the other nucleotides present in the reaction mixture, for example from 2 to 6 times, for instance from 1 to 4 times, such as about 1 times or about 4 times the amount of another nucleotide.
  • the composition contains dUTP as the substitute nucleotide, this is suitably present in an amount of at least 50%, for instance from 1 to 10 times the amount of dTTP present in the composition
  • the ratios of the nucleotides present in an amplification reaction mixture are changed such that a nucleotide is reduced in concentration.
  • This reduction will mean that the nucleotide will be present at a lower concentration than is conventionally used or considered optimal for carrying out an amplification reaction. The reduction is sufficient to slow down the incorporation of the nucleotide into the nascent amplicons.
  • the nucleotide whose concentration is reduced is one which is incorporated early in the primer extension process, in particular the first nucleotide that is incorporated by extension of the primer such as the allele specific primer.
  • the amount or concentration of the said nucleotide is less than 90%, for instance less than 80%, such as less than 70% for example from 20-50% of that or the other nucleotides within the reaction mixture .
  • nucleotides are typically present in a final PCR reaction mixture at concentrations of about 0.5mM,. A reduction of at least the selected one of these nucleotides to a concentration of 0.15mM or less such as 0.12mM or less, for instance from 0.05-0. ImM may enhance specificity.
  • the amounts of all standard nucleotides in the reaction mixture are lowered as compared to that normally used in the amplification reaction.
  • the amount of nucleotides is less than 90%, for instance less than 80%, such as less than 70% for example from 20-50%.
  • nucleotides are typically present in a final PCR reaction mixture at concentrations of about 0.5mM. A reduction of this concentration to concentrations or 0.15mM or less of 0.12mM or less, for instance from 0.05-0. ImM can enhance specificity.
  • the options of the invention may be combined together however.
  • the concentration of standard nucleotides within the reaction mixture may be lower than usual, for instance 0.15mM or less and a substitute nucleotide or modified nucleotide analogue added to the mixture in a relative amount as described above.
  • the substitute nucleotide or modified nucleotide analogue may be present in a range of from 0. ImM to 0.6mM, for instance at 0.15mM also.
  • the amount of the selected one of the nucleotides will be reduced still further.
  • the concentration of the selected one of those nucleotides is suitably no more than 0.09mM, for instance from 0.04 to 0.08mM.
  • the substitute nucleotide may replace all of a corresponding standard nucleotide within the reaction mixture.
  • the reaction mixture may not contain any dTTP.
  • the substitute nucleotide may be present at concentrations of 0.15mM or less.
  • the method may be applied to a variety of nucleic acid amplification reactions where primers are extended, such as PCR and variants thereof such as 'quasa' PCR discussed further below, 'Touchdown PCR' and a range of alternative PCR methods that use different primer(s) and modifications to to enhance specificity. These include but are not limited to, competitive modified primers homologous to the unintended allele (unintended allele specific primer) and 'base clamping' strategies.
  • the invention involves changing the nucleotides in the reaction mixture used to carry out the amplification such as the PCR. This is conveniently achieved by modifying the 'mastermix' which contains all the standard reagents necessary for the reaction.
  • compositions adapted to carry out the methods of the invention including 'mastermixes' that include the basic components (polymerase enzyme, salts such as magnesium chloride, buffers and nucleotides) required in an amplification. These may optionally contain additional elements such as signalling reagents such as fluorescent dyes that may be used in the monitoring of the reaction for quantitation or other purposes.
  • additional elements such as signalling reagents such as fluorescent dyes that may be used in the monitoring of the reaction for quantitation or other purposes.
  • Other compositions will include further yet components such as primers or probes that may optionally be labelled as is known in the art, to allow reactions to be monitored in real-time, again for quantitation purposes. Examples include ScorpionTM, TaqManTM,
  • compositions also include compositions to which a sample has been added, which is ready for amplification.
  • compositions may contain a lower level of at least one nucleotide than is found in conventional amplification reaction mixtures, and/or they may additionally comprise a substitute nucleotide or a modified nucleotide analogue in relative amounts appropriate as discussed above.
  • Such mastermixes are frequently provided in the form of concentrates which are intended for dilution in order to form the final amplication reaction mixtures as described above.
  • the concentration of the nucleotides will generally be much higher in such mastermixes, for instance from 2-10 times more concentrated than in the final reaction mixture, depending upon the level of dilution recommended.
  • the corresponding mastermix will typically have a concentration of from 0.3-1.5mM of that nucleotide for dilution.
  • compositions of this type may be sold alone or as kits including other components required in the amplification reaction, and in particular, allele specific primers as described above. Such kits form a further aspect of the invention.
  • the invention provides a method for preparing a nucleotide composition for use in an amplification reaction mixture, which method comprises either (a) combining together nucleotides for use in the amplification and adding thereto a substitute nucleotide or a modified nucleotide analogue in an concentration sufficient to enhance specificity of an amplification primer for a target sequence; or (b) combining together nucleotides for use in the amplification at differing concentrations so as to enhance specificity of an amplification primer for a target sequence.
  • Novel nucleotide compositions obtainable by this method form yet a further aspect of the invention. These compositions may be added to amplification reaction mixtures as a supply of nucleotides in a 'premixed' form.
  • nucleotide analogues have been added to the reaction mix for allele specific PCR and the applicants have noticed a dose dependant effect in increasing the specificity of the PCR reaction.
  • a wide range of such nucleotide analogues are available and would be known in the art. These include for instance C8-alkyne dCTP , 2-thio-dCTP, 2- aminopurine-drTP and (5-iodo-dUTP).
  • dUTP is a naturally occurring substitute base for dTTP.
  • dUTP is commonly added to a mastermix to render the subsequent PCR product enzymically susceptible to Uracil-N-glycosylase. Treating down-stream PCR reactions with Uracil-N-glycosylase removes potential PCR contamination and is an essential tool in diagnostic PCR. In order to achieve this desirable end the dUTP is used a low concentration typically 5-20% of the level of dTTP. This is because dUTP can reduce the efficiently of amplification.
  • C8- Alkyne-dCTP Another base that has been shown to be useful in the method of the invention is C8- Alkyne-dCTP.
  • This base incorporates as an analogy for dCTP.
  • a wide range of nucleotides analogues within this class and others will be effective in this role as the functional performance of these bases is sequence independent.
  • the essential effect on the allele specific PCR is the same. That is, the performance of the PCR reaction on the authentic template is not affected or is minimally affected whereas the effect of the PCR on a template that has a mismatch base corresponding to the 3 'end of the primer is catastrophic.
  • the addition of analogue bases is therefore an enhancer of the specificity of the allele specific PCR.
  • the increase in sensitivity ranges between 2 and 100 fold.
  • modified nucleotide analogues or substitute nucleotides would be expected to particularly benefit any PCR based system that relies on allele specific PCR.
  • Figure 1 shows the results of an experiment illustrating the addition of dUTP to the quasa assay BRAFV600E available from Primer Design Ltd (UK): 1 X 10E5 copies of wild type or V600E template were amplified with the BRAFV600E quasa assay (SOLID BLACK).
  • SOLID BLACK the BRAFV600E quasa assay
  • the Early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template.
  • the same assay was run including 0.6mM final concentration of dUTP.
  • Figure 2 shows the results of an experiment illustrating the addition of C8-Alkyne-dCTP to BRAFV600E: 1 X 10E5 copies of wild type or V600E template were amplified with the BRAFV600E quasa assay (SOLID BLACK). The Early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template. The same assay was run in the presence of 0.6mM C8-Alkyne-dCTP.
  • Figure 3 shows the results of an experiment illustrating the effect of adding dUTP to the DMNT3A quasa assay available from Primer Design Ltd (UK): 1 X 10E5 copies of wild type or R882H template were amplified with the DNMT3A quasa assay (SOLID BLACK).
  • SOLID BLACK the DNMT3A quasa assay
  • the Early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template.
  • the same assay was run including 0.6mM final concentration of dUTP.
  • Figure 4 shows the results of the an experiment illustrating the effect of adding C8-Alkyne- dCTP to an amplification assay available from Primer Design Ltd (UK) under the name DNMT3 A-R882H: 1 X 10E5 copies of wild type or R882H template were amplified with the BRAFV600E quasa assay (SOLID BLACK).
  • SOLID BLACK BRAFV600E quasa assay
  • the early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template.
  • the same assay was run in the presence of 0.05mM C8-Alkyne-dCTP.
  • Figure 5 shows the results of an experiment illustrating the addition of 5-iodo-dUTP to the quasa assay BRAFV600E available from Primer Design Ltd (UK): 1 X 10E5 copies of wild type or V600E template were amplified with the BRAFV600E quasa assay (SOLID BLACK).
  • SOLID BLACK the BRAFV600E quasa assay
  • the Early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template.
  • the same assay was run including 0.6mM or 0.9 mM final concentration of 5-iodo-dUTP
  • Figure 6 shows the results of an experiment illustrating the addition of 2-aminopurine-drTP to the quasa assay BRAFV600E available from Primer Design Ltd (UK): 1 X 10E5 copies of wild type or V600E template were amplified with the BRAFV600E quasa assay (SOLID BLACK).
  • SOLID BLACK BRAFV600E quasa assay
  • the Early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template.
  • the same assay was run including 0.6mM or 0.9 mM final concentration of 2-aminopurine-drTP
  • Figure 7 shows the results of an experiment illustrating the addition of 2-Thio-dCTP to the quasa assay BRAFV600E available from Primer Design Ltd (UK): 1 X 10E5 copies of wild type or V600E template were amplified with the BRAFV600E quasa assay (SOLID BLACK).
  • SOLID BLACK the BRAFV600E quasa assay
  • the Early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template.
  • the same assay was run including 0.6mM or 0.9 mM final concentration of 2-Thio-dCTP.
  • Figure 8 shows the results of an experiment illustrating the effect of reducing the amount of a nucleotide within an allele specific amplification reaction on the DNMT3A-R882H assay.
  • the early CT value detection represents authentic detection on the mutant template whist the later trace is the non-specific detection on the wild type template.
  • the same assay was run at a reduced dGTP concentration of 0.05mM final concentration
  • Example 1
  • a particular embodiment for the method of the invention is to use it in conjunction with 'quasa' (Quantitative allele specific amplification) system available from Primer Design Ltd (UK).
  • QuasaTM is a variation on allele specific PCR that uses simple manipulations of primer design in conjunction with modified thermo-cycling parameters to achieve a high level of both sensitivity and specificity.
  • Quasa primer have an exceptionally low Tm (40-50°C) and also include 5' sequence independent tag.
  • Quasa cycling parameters occur in two stages.
  • the first set of cycling parameters have an exceptionally low annealing temperature (50-55°C). This enables the very low Tm allele specific primer to prime efficiently on the target template.
  • the second set of thermo-cycling parameters have a much higher annealing temperature (60°C). These cycles permit efficient PCR amplification of templates that have already incorporated the allele specific primer including the 5 'tag which increases the effective Tm of the primer for nascent cDNA. At this higher temperature, further priming on the input target sequence is prevented.
  • BRAF-V600E and DN MT3A-R882C are mutations of clinical significant in a range of cancers and leukaemia.
  • Amplicons corresponding to both the mutant and wild type amplicons were cloned using commercial services.
  • the cloned amplicons were linearised and prepared at precise copy number dilutions (1 xlO 5 ).
  • Mastermix was prepared using goTaq from Promega which uses native taq DNA polymerase in a proprietary buffer. Each reaction contained 5 X goTaq reaction buffer, 0.3U of enzyme and MgCl 2 at a final concentration of 4mM and a dNTPs at a final concentration of 0.15mM. This mix was supplemented with dUTP, C8-Alkyne-dCTP, 5-iodo-dUTP, 2-Thio-dCTP or 2- aminopurine-drTP at a range of final concentrations from O. lmM to 0.9mM.
  • the DNMT3A-R882C assay as described above was repeated using a mastermix comprising reduced levels of dATP, dCTP and dTTP and dGTP to a concentration of 0.15mM. In a separate well, the concentration of dGTP was reduced further to just 0.05mM on both wild- type and mutant template.

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Abstract

L'invention concerne un procédé permettant de mettre en œuvre une réaction d'amplification d'acide nucléique pour amplifier une séquence spécifique qui comprend un polymorphisme, ledit procédé consistant à mettre en œuvre une réaction d'amplification d'acide nucléique à l'aide d'un mélange réactionnel d'amplification d'acide nucléique qui comprend une amorce d'acide nucléique ayant une extrémité 3' qui se lie au niveau ou près du site de polymorphisme, et un ensemble de nucléotides apte à effectuer l'amplification de la séquence cible et la composition des nucléotides étant choisie de façon à améliorer la spécificité de l'amorce de la séquence spécifique. En particulier, un substitut nucléotide ou un analogue de nucléotide modifié qui entre en compétition avec le nucléotide naturel pour incorporation est inclus dans la réaction à une concentration relativement élevée, et/ou la concentration d'un ou plusieurs des nucléotides est réduite.
PCT/GB2014/050102 2013-02-06 2014-01-15 Technique d'amplification WO2014122422A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB1302112.6 2013-02-06
GBGB1302112.6A GB201302112D0 (en) 2013-02-06 2013-02-06 Amplification technique
GB1308291.2 2013-05-08
GB201308291A GB201308291D0 (en) 2013-05-08 2013-05-08 Amplification technique

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WO2014122422A2 true WO2014122422A2 (fr) 2014-08-14
WO2014122422A3 WO2014122422A3 (fr) 2014-11-27

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN104389026A (zh) * 2014-10-30 2015-03-04 北京诺禾致源生物信息科技有限公司 单细胞转录组测序文库的构建方法及其应用
WO2019077338A1 (fr) 2017-10-17 2019-04-25 Hd3 Limited Dispositif de coiffure, procédé de coiffure et système d'entraînement

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WO2009151921A1 (fr) * 2008-05-27 2009-12-17 Trilink Biotechnologies Nucléosides 5’-triphosphates modifiés chimiquement pour l’amplification initiée thermiquement d’un acide nucléique

Patent Citations (1)

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WO2009151921A1 (fr) * 2008-05-27 2009-12-17 Trilink Biotechnologies Nucléosides 5’-triphosphates modifiés chimiquement pour l’amplification initiée thermiquement d’un acide nucléique

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"BRAF V600E Handbook" In: "BRAF V600E Handbook", 15 January 2004 (2004-01-15), Primer design Ltd, XP055143531, the whole document *
PASTINEN T ET AL: "A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays", GENOME RESEARCH, COLD SPRING HARBOR LABORATORY PRESS, WOODBURY, NY, US, vol. 10, no. 7, 1 July 2000 (2000-07-01), pages 1031-1042, XP002228091, ISSN: 1088-9051, DOI: 10.1101/GR.10.7.1031 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104389026A (zh) * 2014-10-30 2015-03-04 北京诺禾致源生物信息科技有限公司 单细胞转录组测序文库的构建方法及其应用
WO2019077338A1 (fr) 2017-10-17 2019-04-25 Hd3 Limited Dispositif de coiffure, procédé de coiffure et système d'entraînement
EP3977890A1 (fr) 2017-10-17 2022-04-06 HD3 Limited Dispositif de coiffage, procédé de coiffage et système de commande

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