EP0286642A1 - Procede d'analyse de polynucleotides base sur le deplacement des brins, et complexe reactif - Google Patents

Procede d'analyse de polynucleotides base sur le deplacement des brins, et complexe reactif

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Publication number
EP0286642A1
EP0286642A1 EP87900593A EP87900593A EP0286642A1 EP 0286642 A1 EP0286642 A1 EP 0286642A1 EP 87900593 A EP87900593 A EP 87900593A EP 87900593 A EP87900593 A EP 87900593A EP 0286642 A1 EP0286642 A1 EP 0286642A1
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
reagent
binding region
target
labeled
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Withdrawn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
EP87900593A
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German (de)
English (en)
Other versions
EP0286642A4 (fr
Inventor
Mary Collins
Joseph P. Dougherty
Edward Francis Fritsch
Kenneth A. Jacobs
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.)
Genetics Institute LLC
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Genetics Institute LLC
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Publication date
Priority claimed from US06/809,992 external-priority patent/US4752566A/en
Application filed by Genetics Institute LLC filed Critical Genetics Institute LLC
Publication of EP0286642A1 publication Critical patent/EP0286642A1/fr
Publication of EP0286642A4 publication Critical patent/EP0286642A4/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to a diagnostic assay method for detecting the presence of a target nucleotide sequence (either DNA or RNA) in a biological sample, a polynucleotide reagent complex therefor and diagnostic kits therefor.
  • a target nucleotide sequence either DNA or RNA
  • a labeled polynucleotide or signal strand bound in a reagent complex to the probe polynucleotide generally by complementary base pair binding to a portion of the target binding region.
  • the portion of the labeled polynucleotide so-bound is referred to as the pairing segment.
  • the target nucleotide sequence binds to the target binding region and displaces the labeled polynucleotide.
  • the displaced labeled polynucleotide is then detected as a measure of the presence and amount of target nucleotide sequence in the sample. While in such systems, the probe and labeled polynucleotide are generally
  • Two forms ' of separationof displaced labeledpolynucleo- tides from intact reagent complexes involve: (a) a reagent complex in which the probe polynucleotide is immobilized and (b) a reagent complex in which the probe polynucleotide is in solution with a pendant affinity moiety (e.g. biotin) for post-displacement immobilization.
  • a pendant affinity moiety e.g. biotin
  • tolerable background will depend upon both the number of reagent complexes used in the reaction and the amount of analyte to be detected. For example, to detect 10 s analytes in a reaction with 10 11 reagent complexes, 0.001% (10 6 ) or fewer complexes must be released or not immobilized as background for any signal to be detected with a [signal plus background] background ratio of at least 2:1.
  • EPA 167,238 and 164,876 embodiments are shown (e.g., in Figure 2A) wherein the labeled polynucleotide L bears an affinity moiety (biotin) for post displacement immobilization. While such technique concentrates the signal, it is unlikely to aid in background reduction, since displaced labeled polynucleotides and detached reagent complexes each have biotin and are likely to be immobilized with substantially equal efficiency. (See also, EPA 200 , 057 ) .
  • biotin affinity moiety
  • the methods and reagents of the present invention involve diagnostic assays which may be used for detecting and determining the presence and/or concentration of an infectious agent or for distinguishing the strain, regulatory state or other state (including drug resistances) of an organism or vector.
  • the methods of the invention are improved methods for carrying out strand displacement assays including the assays described in EPA 167,238 and 164,876.
  • the present invention provides a method (“Method I”) for determining the presence of a predetermined target nucleotide sequence (“TNS”) in a biological sample.
  • This method includes the following steps: First, an inverse reagent complex (“IRC")is formed of (i) labeled probe polynucleotide (“labeled probe") having a target binding region (“TBR”) which is capable of complementary base pair binding via hydrogen bonds of purine/pyrimidine bases to the target.
  • IRC inverse reagent complex
  • labeled probe polynucleotide labeled probe polynucleotide
  • TBR target binding region
  • Another portion of the complex is a second polynucleotide which is bound to the labeled probe in a region of the labeled probe at least partially coextensive with the TBR.
  • the IRC is contacted with a sample under conditions in which the target, if present, binds to the labeled probe and displaces second polynucleotide therefrom.
  • the labeled probes from which second polynucleotides have been displaced are separated from intact IRCs. Finally, the presence and/or amount of labeled probes which have been separated are determined.
  • the present invention provides a diagnostic reagent, the IRC, for use in Method I.
  • the IRC includes the labeled probe and second polynucleotide as described above.
  • the second polynucleotide can be immobilized or immobilizable.
  • the potential binding between the target and the labeled probe is capable of displacing the second polynucleotide from the labeled probe.
  • the present invention provides another diagnostic reagent, the inverse reagent complex construct ("IRCC”) for use in Method I.
  • the IRCC includes a single polynucleotide strand having (i) a TBR capable of binding to the target; and (ii) a pairing segment ("PS”) bound to a portion of the TBR.
  • a detectable tag which is within or adjacent to the TBR.
  • the present invention provides a method (“Method II”) for determining the presence and/or amount of a predetermined target in a biological sample.
  • a reagent complex is formed of a probe polynucleo ⁇ tide (hereafter “probe") which has a TBR and a labeled polynucleotide (hereafter “label strand”) bound to the probe in a region of the probe at least partially coextensive with the TBR.
  • probe probe polynucleo ⁇ tide
  • label strand labeled polynucleotide
  • the reagent complex is then contacted with a sample and with a capture polynucleotide ("CP") under conditions in which the target, if present, binds to the probe displacing label strand from the reagent complex.
  • the CP binds selectively to the displaced label strand in a pairing segment ("PS"), i.e. the region thereof that had been bound to the probe.
  • PS pairing segment
  • Displaced label strand which has bound to CP is then separated from unbound label strand. The captured displaced label strand is then detected.
  • the probe polynucleotide and label strand of the reagent complex are parts of a single polynucleotide strand folded onto itself.
  • This single strand may also include two or more stably joined polynucleotide strands, e.g., joined by covalent or non- covalent bonding stable to the conditions of hybridization and displacement.
  • the label strand includes a PS with a tag and the probe includes a TBR.
  • a hairpin loop may be present which divides the label strand portion from the probe portion of the single strand. [See Figs. 4A to 4D].
  • This reagent complex is referred to as the reagent complex construct (RCC) .
  • the invention provides a kit for determining the presence and/or amount of target in a sample.
  • the kit includes a reagent complex formed of the above-described probe and a label strand bound through its PS to a label strand binding region of the probe ("PS"').
  • PS 1 is at least partially coextensive with the TBR.
  • a CP having a second binding region capable of binding selectively to a segment of displaced label strand substantially within the ps (hereafter "CS'") is also part of this kit.
  • the third component of the it is a means for isolating the CP together with any attached displaced label strand from unbound label strand.
  • the kit contains an RCC and a detectable tag attached within or adjacent to the TBR.
  • Method III a method for use in diagnostic tests for disrupting the linkage between reagents which are attached by binding through the highly stable biotin-avidin or biotin- streptavidin bond.
  • Method III the attached reagents are incubated in the presence of excess biotin at moderate temperatures ranging from 22-65 C.
  • Method III can be used in a tetraethylammonium chloride (“TEAC1”) salt solution.
  • TEAC1 tetraethylammonium chloride
  • the lower temperature or the combination of lower temperature and of TEAC1 allows the use of Method III with heat labile substances such as enzymes, antibodies, antigens and other proteins in formats compatible with DNA hybridization.
  • antibody-antigen binding may be used in Method III in the area of both DNA diagnostics and immunodiagnostics where enzymes ' or other signals are attached to either DNA molecules, antigens or antibodies through an avidin-biotin linkage, or where reagents in a diagnostic test are attached to a support or surface through a biotin-avidin linkage. Additional uses include purification schemes utilizing binding to substances linked to a support through a biotin-avidin linkage. [See, e.g. Hofman et al, Biochem. 2.1:978-984 (1982)].
  • Method III The displacement of Method III by dissolved biotin at moderate (22-65 degrees C) temperatures has significant and unexpected advantages over the prior art.
  • T. Kempe et al. Nucleic Acids Res., 13:45-47 (1985) teaches treatment by near boiling (90 degree C) for five minutes. At these high temperatures melting of double-stranded DNA occurs and enzymes and other proteins denature.
  • Method III allows the biotin-displacement of strands having both biotin and label under conditions suitable for releasing the immobilized complexes of CP and labeled polynucleotide or of CP and complex hybridized to target.
  • the methods and kits of the present invention are capable of reducing background label detection in displacement assays where the proportion of displaced label relative to intact reagent complexes is low and an amount of background label detection is attributable to detached reagent complexes, cleaved reagent complexes and/or intact reagent complexes not immobilized in a postdisplacement immobilization step.
  • Such methods and kits are further suited to certain embodiments of strand displacement in solution in which the CP is the sole or primary means for separating displaced label strand from intact reagent complexes.
  • Fig. 1 is an embodiment of an IRC (A) onto which sample polynucleotide G having target nucleotide sequence TNS has hybridized (B) r and ultimately completely displaces PS of second polynucleotide SP from labeled probe LP (C) .
  • Fig. 2 illustrates an IRCC (A) , which has been contacted by G (B) and which becomes bound by immobilized CP (C) .
  • Fig. 3 illustrates two immobilized reagent complexes on a support (A) ; sample G causing displacement of label strand LI (B) ; G bound to probe PI and LI in solution with immobilized CP having a capture segment CS (C) and finally LI immobilized on CP (D) .
  • Fig. 4 illustrates RCC (A) ; G binding to RCC and displacing PS (B) ; a biotinylated CP (C) , which hybridizes to RCC (D) and an intermediate structure of a hybrid between G and CP (E).
  • Fig. 5 illustrates a covalent complex p66d and CPs therefor.
  • TNS is the target nucleotide sequence or analyte in a biological sample.
  • Labelled probe is the labelled polynucleotide probe employed in Method I.
  • Probe is the polynucleotide probe of Method II.
  • TBR is the target binding region of the labelled probe of Method I and the probe of Method II.
  • IBR is the initial binding region, which is a single stranded portion of the TBR to which TNS can bind without displacing the second polynucleotide of Method I or label strand of Method II.
  • Second polynucleotide refers to the polynucleotide strand of Method I which binds to the labelled probe.
  • PS is the pairing segment portion on the second polynucleotide and label strand, whjich binds to the TBR on the labelled probe or probe, respectively.
  • PS 1 is the region of the labelled probe or probe usually located in the TBR, which binds to the PS.
  • CP is the capture polynucleotide strand of Method II.
  • CS is the region of CP capable of binding a portion of the second polynucleotide or label strand.
  • CS• is the portion of the second polynucleotide or label strand capable of binding to CS and located in the PS.
  • RCC refers to a reagent complex construct.
  • IRC refers to- an inverse reagent complex of Method I.
  • IRCC refers to an inverse reagent complex construct used in
  • Method I The basic components of Method I are a labeled probe, a second polynucleotide and a biological sample containing nucleic acid, a portion of which is TNS.
  • Other optional components of this method include a volume-excluding polymer, such as a polyether compound
  • a support to which the reagent complex is immobilized via the second polynucleotide may be a component of the method.
  • a support may also be employed as part of the separation step that follows displacement.
  • additional reagents or equipment may be required for "readout". "Readout" is the direct or indirect detection of labeled probe in one or more phases of separated reaction materials.
  • Readout reagents may particularly be needed in the liquid phase by virtue of the displacement of the IRC and separation of labeled probe fromwhich second polynucleo ⁇ tide has been displaced from the solid phase containing immobilized displaced second polynucleotides and immobilized intact IRC.
  • Method II The basic components of Method II include a CP, a probe, a labeled polynucleotide and a biological sample containing TNS.
  • “Capturing” refers to hybridization of the displaced label strand to a CP which is at least partially complementary to the label strand in a region of the label strand substantially or completely within the PS that had been bound to the TBR in the reagent complex. PS is thus available for hybridization to the CP only when it has been displaced from the probe TBR.
  • the probe employed in the present invention may be a linear or circular polynucleotide (RNA or DNA) capable of binding in at least one region of its purine/pyrimidine base sequence to specific TNS of a sample.
  • TNS which can be analyzed include viral or bacterial nucleic acid sequences, DNA or RNA sequences of plants, animals (including humans) or microorganisms (including plasmids) and rRNA and other non-protein coding sequences.
  • binding may be between DNA and RNA, between DNA and DNA or between RNA and RNA. It is generally only a specific region of the probe which binds selectively to the TNS.
  • regions of the probe may be various naturally occurring or synthesized sequences, which do not participate in the hybridization reaction with the target, but which may play an important role in the present invention, e.g., by serving as a site for attachment to a label or by providing some degree of separation between the label and the region to which the target binds, if desired. Sizes, types and geometries of probe, TBR, second polynucleotide and label strand may be as set forth in the published patent applications referred to herein.
  • the region of the probe to which TNS will specifically bind is the TBR.
  • the binding at the TBR may be, and preferably is, perfect.
  • Each nucleotide in the probe finds its correct complementary binding partner (e.g., dA to dT) in TNS.
  • the hybridization may contain some mismatches, as described in the referenced published applications.
  • At least one portion of the TBR is preferably single-stranded (i.e., it is not complementary to second polynucleotide sequences nor self-complementary) .
  • This single-stranded region is called the initial binding region ("IBR") , because TNS can bind to this region of bases without displacing any of the second polynucleotide.
  • the IBR of the probe is at least fifteen bases in length, and is preferably at least fifty bases in length.
  • the complete TBR includes the IBR and preferably all of the PS• , the region which binds to the second polynucleo ⁇ tide or label strand.
  • the complete TBR may include less than the complete PS' . While such pairing to the second polynucleotide or label strand is normally confined to a portion of the TBR, some limited number of base pairs outside of the TBR (e.g. , up to 15 such nucleotide pairs) is permitted, but not preferred. The effect of such an overhang or residual binding region corresponds to the effect of the RBR discussed in relation to Fig. IG of EPA 167,238.
  • the length of the complete TBR is not independently critical, but can be considered a function or sum of the preferable lengths of the IBR and PS 1 portions.
  • Base lengths of the IBR above five hundred are generally not required but can be used.
  • a suitable lower limit on the length of the IBR depends upon the base sequence of the TBR and base composition and other physical factors, including the conditions and kinetics of the intended hybridization, and the readout system employed.
  • the probe also contains a detectable tag.
  • One or more tags may be located, using conventional techniques, at one of several points along the labeled probe, especially if the tag is a radionu ⁇ lide or biotin or the like.
  • one or more tags may be located only at one end or only at one specific internal location on the labeled probe (e.g., at a purine or pyri idine base not involved in base pairing to the second polynucleotide) .
  • the tag is preferably located or concentrated in a probe region outside of the TBR.
  • detectable tags especially if remote from the probe TBR, should have little or no effect on the strength of base pairing between the labeled probe and either the second polynucleotide or TNS. In practice this may be determined by little or no diminution of the IRC melting temperature and, more importantly, by negligible effects on the hybridization reaction between TNS and the labeled probe.
  • the second polynucleotide or label strand (DNA or RNA) includes a pairing segment PS bound in the reagent complex to the labeled probe or probe.
  • the length of such PS corresponds to the length of the probe PS' , mentioned above.
  • the pairing between the second polynucleotide and the labeled probe or the label strand and the probe can be perfect or can include a limited number of mismatches.
  • the second polynucleotide or label strand can also be bound in the reagent complex via multiple PS, each to a portion of a unique TBR of the labeled probe.
  • Such second polynucleotide or label strand would be fully displaced either by multiple targets of the sample, or by one or more sample targets and one or more selected nucleotide sequences of a reagent polynucleotide. Additionally, the selected reagent polynucleotide may bind to the second polynucleotide to displace a PS from a portion of a TBR in an inverse fashion.
  • the second polynucleotide can also contain a moiety capable of achieving physical separation of labeledprobes fromwhich secondpolynucleotide has been displaced from intact IRC (e.g. through immobilization, size separation, phase separation, gel electrophoresis and the like) .
  • a physical separating moiety is exemplified by biotin, wherein the post- displacement separation would involve immobilized avidin, streptavidin or anti-biotin antibody.
  • the second polynucleotide can be immobilized by a solid support in the IRC.
  • the physical separating moiety and the act of physical separation shall be referred to as "immobilizable”. Covalent linkages are preferred when second polynucleotides are immobilized to a solid support in the IRC.
  • the CP In addition to containing the sequence CS, the CP also should have some property, or modification to make this capturer separable from intact labeled or reagent complexes as well as from other sources of background, such as detached tags. Suchmodifications include attachment of the CP to a solid support, such as colloidal particles, filters or membranes, and attachment of affinity reagents such as biotin to the CP. Alternatively, the CP can be significantly larger than the unreacted displacement complex to allow a size separation after hybridization.
  • Method II is applicableto displacement assays performed both on a solid support and solely in solution.
  • capturing reduces background signal in the assay which may result from the detachment of unreacted reagent complexes from the support due to breakage of the polynucleotide, failure of the attachment reaction, degradation of the solid support or other sources of non- specifically detached label.
  • a second benefit is that the captured label strand can be removed from the displacement reaction and transferred to a position or condition which is more advantageous for readout.
  • the combination of solid support displacement reactions with the use of CP has the advantage of potentially higher signal to noise ratios, due to two separation steps, and may be simpler to use.
  • capturing provides a method for separating displaced label strand from unreacted reagent complexes. Captured label strands from solution assays can also be transferred to more advantageous positions for readout.
  • Solution displacement reactions have the potential advantage of better hybridization kinetics than reactions on solid supports, and allow the use of polymers such as polyethylene glycol (PEG) to enhance these reaction rates.
  • PEG polyethylene glycol
  • a CP bearing an affinity reagent such as biotin permits both the initial displacement reaction and the capturing reaction to be carried out in solution. Separation of the displaced probe is achieved by trapping captured label strand on an avidin support (alternatively, an immobilized streptavidin or anti-biotin antibody) .
  • An additional advantage is that the displacement reaction can be carried out using an easily prepared complex which contains the label strand and TNS in a single polynucleo ⁇ tide strand.
  • This "covalent displacement complex" (RCC) simply unfolds after hybridization and strand displacement by the target, making the complement of the CP (CS' in Figure 4B hereof) single stranded and available for hybridization. Only RCC which have hybridized to TNS can be captured.
  • the CP can be added after the displacement reaction has been completed, or can be present during the displace ⁇ ment reaction.
  • the concentration of CP will be greater than the concentration of target in the sample and can be greater or smaller than the concentration of reagent complexes. Since the target and CP will be complementary over at least a portion of each sequence, target can first hybridize to the CP (Fig 4C) . Since the target is still available for hybridization to the TBR of the reagent complex, this CP-target hybrid (the "first intermediate complex”) can now hybridize to the labeled reagent complex orming a "second intermediate complex". The second complex may be stable in this configuration, or may resolve by strand exchange and be recaptured.
  • the displacement complex will only be captured when it has hybridized to target. If, however, second intermediate complexes are assayed as positive, then some of the characteristics of a sandwich assay are imparted. Therefore, some sample polynucleotides may be considered targetwhichhave regions complementaryto differentportions of the TBR spaced too far apart to cause displacement with high efficiency or which are incapable of complete displacement for a variety of reasons.
  • the capturing reaction is compatible with many readouts including enzymes, polyA tails (see EPA 200,057 and 200,056) fluorescent latex beads and enzyme substrates.
  • capturing can occur in a defined space on a surface when the CP is already attached to a solid support.
  • label strand can be trapped at a particular position on a surface coated with avidin.
  • An avidin coated "dipstick” could be used to trap captured label strand.
  • avidin could be attached to surfaces to allow trapping in a predefined position for automated signal readout.
  • the CP provides the only means for separation (or the primary means if other steps are included) , and must bind to displaced label strand with high selectivity (not binding to undisplaced reagent complexes) . If the CP is immobilized or immobilizable onto a support, then the nonspecific binding of intact or broken reagent complexes to the support must be rare.
  • the fraction of nondisplaced reagent complexes adhering to the support, either through nonspecific capturing or nonspecific binding to the support that would be per issable in the reaction is a function of both the initial number of reagent complexes and the number of target molecules to be detected. The relationship between these parameters can be described as follows.
  • 10 ⁇ target molecules can only be detected with a (signal and background)/background ratio of at least 2/1 if background nonspecific binding is less than 1%.
  • the signal to noise ratio increases, with a resulting increase in assay sensitivity.
  • capture alone can provide adequate specificity to a solution displacement assay.
  • capturing can be combined with both probe immobilization and/or release of captured tag from a support.
  • Directly detectable tags for use in the methods, kits and reagents of the present invention include radionuclides and fluorescein compounds commonly employed in diagnostic probes attached to the free end of a labeled probe or to one or more of the bases of a labeled probe. Moieties directly detectable by other means including being cleaved off, being detectable ⁇ olorimetri ⁇ ally or otherwise, like nitrophenol, may also be used as tags.
  • Indirectly detectable tags for use in the invention include those modifications that can serve as antigenic determinants, affinity ligands, antigens or antibodies recognizable through immunochemi ⁇ al or other affinity reactions, such as described inpublishedpatent applications EPA 63,879, WO83/02277, and EPA 97,373.
  • Other such tags include biotinylated nucleotides present in or added onto the labeled probe (e.g., by the enzyme terminal deoxynucleotidyl transferase, which will add multiple nucleotides at the 3 ' end of the labeled probe in the absence of a template strand) .
  • tags are enzymes attached to a labeled probe (especially at a free end remote from the TBR) whose presence can be determined after the displacement and separation steps of the methods by addition of the substrate for the enzyme and quantifi ⁇ cation of either the enzymatic substrate or, preferably, the enzymatic reaction product.
  • the tag may be an apoenzyme, co-enzyme, enzymatic modifier, enzymatic cofactor or the like, with the other necessary reagents usually added after displacement and separation, along with the appropriate enzymatic substrate. If the enzymatic reaction cannot occur with all but one component present (e.g., the substrate), these other reagents may be present in solution during the contacting or displacement steps.
  • ribonucleotide segment tags described in European patent applications 200,056 and 200,057.
  • a polyriboadenosine -("poly A") segment may be formed on the 3' end of a DNA portion of the probe including the TBR.
  • the poly A segment can be digested with polynucleotide phosphorylase to riboadenosine diphosphate, which can then be phosphorylated by phosphoenol pyruvate in the presence of pyruvate kinase to adenosine triphosphate ("ATP”) .
  • This ATP can then be detected (e.g. , with luciferin/luciferase) as an amplified signal indicative of the number of such labeled probes so separated.
  • Multiple detectable tags can be added in the process of manufacturing the labeled probe or label strand of these methods by enzymes, such as terminal deoxynucleotidyl transferase, DNA ligase, polynucleotide phosphorylase, and the like. Multiple labeled probes or strands, each containing a signalling moiety or detectable tag, can also be used.
  • One form of attachment of an enzyme to the labeled polynucleotide is via affinity reagents, e.g, streptavidin to biotin. Such a binding form can be used in various embodiments, (e.g.
  • the complex is prepared by hybridizing a biotin-labeled polynucleotide to the probe and then binding a streptavidin-enzyme conjugate to the biotin prior to the contacting/displacement step described above. Additionally, a moiety interacting with the detectable tag in the complex may be present on the second polynucleotide.
  • the tags have been illustrated in the Figs, as on an end of a nucleic acid strand. However, other forms of single or multiple tags incorporated in or on the appropriate polynucleotide segment or strand can be used.
  • the important features of the tag are that it be directly or indirectly detectable. Additionally, for Method II, it is important that the tag be positioned or attached to remain after displacement and capture with the appropriate strand or segment (PS in most cases) .
  • Method III involves disrupting the affinity linkage between biotin and avidin or biotin and streptavidin in a manner that avoids denaturing or disrupting the binding forces associated with diagnostic reagents attached to these affinity linkers.
  • This method of disrupting the biotin/avidin linkage is particularly adaptable to Method II herein, where the CP is immobilized by such a linkage.
  • Method III is a method for releasing a diagnostic reagent bound to a- support (or other substance) by a biotin/avidin or biotin/streptavidin linkage comprising incubating the bound diagnostic agent in the presence of an excess concentration of free biotin to the concentration of avidin or streptavidin present in the bound reagent-support complex.
  • the biotin concentration can be lmM or greater.
  • Method III is most preferable where the reagent or support is singly biptinylated. The incubation is performed at temperatures of between 22 to 65 degrees centigrade. The time required for the release is dependant on the temperature employed.
  • the diagnostic reagent and support may be similar or different entities, and may include single or double stranded DNA or RNA, individual nucleic acids, oligo or polynucleotides, hybrids thereof, enzymes, cofactors, antigen, antibody, signalling or labelling moieties, tags, and derivatives and modifications thereof.
  • the support can be a "standard" support such as latex, agarose, filter, membrane, or natural, synthetic or semi-synthetic polymers.
  • the reagent is a double stranded polynucleotide, which remains undenatured by the disrupting step.
  • the reagent may be a biotinylated nucleic acid and the support is an avidin or streptavidin-associated nucleic acid.
  • the diagnostic reagent of Method III can be a capture strand.
  • one reagent is a biotinylated DNA and the support is avidin conjugated to a signalling moiety, such as an avidin-enzyme.
  • the reagent can be a biotinylated antigen and the support an avidin-antibody.
  • Figs. 1A through 1C illustrate one embodiment of Method I, a displacement assay employing the IRC according to the present invention.
  • Second polynucleotide (SP) is immobilized at one end by attachment to a support (SU) and has a PS at the opposite end.
  • the labeled probe (LP) has a detectable tag (T) at one end and a TBR including about half of the complete length of the LP, including the end opposite the T. PS' of TBR is bound to PS of SP.
  • the interior half of TBR is a single-stranded IBR.
  • IB illustrates the IRC after a sample polynucleotide (G) having the target (TNS) has hybridized to the IBR.
  • G sample polynucleotide
  • TMS target
  • IBR' portion complementary to IBR
  • IBR/IBR 1 double-stranded portion
  • Branch migration may now occur in which PS and the remainder of TNS zip and unzip within the end half of TBR. As described in EPA 167,238 this phenomenon is very rapid. Within several minutes or less, the displacable strand (in this illustration, SP) is displaced. As shown in Fig. 1C, TNS has now completely displaced the PS of the second polynucleotide from the TBR of the labeled probe. The hybrid G/LP which includes a tag T can now migrate away from the support and be separated therefrom in a liquid phase. All intact IRCs which have not been contacted by TNS should remain on support SU, as illustrated in Fig. 1C.
  • the tags detected directly or indirectly in the separated liquid phase give a qualitative and a quantitative measure of the TNS in the sample.
  • Departures from the geometry of Figs. 1A to 1C within the scope of the invention can be understood by reference to various Figs of the strand displacement assay of EPA 167,238.
  • the second polynucleotide PS binds only to a portion of TBR. There is no residual binding region of labeled probe bound to PS but not part of TBR. Some number of such nucleotides are permissible; but in general such a residual binding region is preferably no greater than 15 nucleotides in length. [See Fig IG and Example 13 of EPA 167,238].
  • Such a residual binding region is present at the stage of complete displacement (Fig. 1C) wherein TNS is fully hybridized with TBR, such residual binding region of the probe could still be bound to PS.
  • the reaction conditions e.g., temperature and salt concentration
  • the end of the second polynucleotide opposite to PS has a moiety (e.g., biotin, as in a poly-bio-d ⁇ tail) immobilizable by an immobilized avidin or streptavidin.
  • the displacement is preferably conducted in solution.
  • the reactionmixture is contactedwithimmobilized avidin or streptavidin to immobilize intact IRCs and displaced second polynucleotides.
  • Sample strand/labeled probe hybrids are left in solution for subsequent direct or indirect detection.
  • a homopolynu ⁇ leotide tail e.g., poly-dC
  • the post-displacement immobilization step would employ an immobilized complementary homopolynucleotide (e.g, oligo- dG-cellulose to immobilize the poly-dC tails of SP) .
  • the labeled probe/sample polynucleotide hybrid is in a liquid phase for subsequent detection, intermediate treatments can be conducted to obtain further information (e.g., restriction fragment size or sequence information) about sample polynucleotide.
  • the liquid phase may be subjected to gel ele ⁇ trophoresis [See, S. G. Fisher and L. S. Lerman, Proc. Natl. Acad. Sci., USA. 72, 989-993 (1983); R. M. Myers et al, Nature, 313: 495-498 (1985)], restriction endonuclease treatment, SI nucleasedigestion or ribonuclease digestion [R. M. Myers, et al.. Science. 230: 1242-1246 (1985)] to develop information beyond merely the number of target sequences.
  • a special embodiment of the diagnostic reagents of the present invention is the IRCC of Fig. 2A, which has a single polynucleotide strand including a PS near one end and a TBR adjacent to the opposite end. This opposite end also bears a detectable tag T.
  • PS (containing a portion labelled CS') . is bound by complementary base pairing to the half of the TBR nearest to T, leaving the interior half of the TBR single-stranded as an IBR.
  • Fig. 2B an IRCC has been contacted by a sample polynucleotide strand G containing the target TNS. TNS has bound to all of the TBR, displacing PS.
  • PS is in single-stranded form, remaining attached to TBR and T by the covalent phosphate/sugar backbone. Alternatively, other stable attachments could be employed for this purpose.
  • Fig. 2B The hybrid of Fig. 2B is illustrated in Fig. 2C as subsequently bound by an immobilized CP which contains a portion CS complementary to a portion on the IRCC, designated CS' .
  • CP will capture the hybrid, but not the intact IRCC of Fig. 2A.
  • CP can also hybridize to sample polynucleotide G directly, forming an intermediate (CP-G) which can then hybridize to the IRCC probe complex.
  • intermediates can function in the assay, it may be desirable in using the IRCCs of Fig. 2A, to employ proportions, orders of addition or other parameters either to avoid or to fully resolve any such intermediate structures as may form.
  • IRCC Fig.2Awhereby the IRCC is initially immobilized.
  • Initial immobilization of the IRCC requires an immobilized third polynucleotide having a binding segment complementary to and bound to a portion of TBR other than the portion of TBR bound to PS.
  • binding segment would be bound to a portion of the IBR segment shown in Fig 2A or alternatively, the TBR would extend to the right of the PS' portion and the binding segment would be bound to all or a portion of TBR to the right of PS* .
  • Displacement of such third polynucleotide would release IRCC from the support.
  • the tag T can be detected on the solid phase or can be introduced into a new liquid phase after the liquid phase containing intact IRCCs has been removed.
  • Figs. 3A through 5 illustrate Method II.
  • Fig. 3A shows two reagent complexes on a support, having probes Px and P 2 with identical TBR.
  • a label strand or signal strand L and L 2 ) is bound to a portion of each TBR.
  • the portion of L ] _ so-bound is PS.
  • the portion of TBR not bound to PS is the IBR.
  • sample strand G containing TNS has bound to the IBR of P ⁇ « The reagent complex including probe P 2 is intact.
  • the top reagent complex is subject to strand migration and ultimately displacement of segment PS from TBR. The result, shown on the left of
  • Fig. 3C is that G is bound to P ⁇ , via TNS and TBR and that
  • a portion CS* of the PS of the label strand is shown.
  • CP is shown in immobilized form, with a capture segment CS which is complementary to segment CS 1 of L ⁇ . After the displaced L- ⁇ is in solution, it can hybridize to CP via CS'/CS, forming the captured structure shown in Fig. 3D.
  • Fig. 4A illustrates a RCC having a TBR near one end and a PS near the other end, with a tag T on the end near PS.
  • PS is hybridized to the end-most portion of TBR, leaving the interior portion IBR of TBR single-stranded. No optional hairpin structure separating TBR from PS is shown in Fig. 4A.
  • Fig. 4B a sample polynucleotide G having a TNS has bound to IBR (see Fig. 4A) and displaced PS from TBR, forming a complete TNS/TBR double-stranded segment.
  • the PS, adjacent to the tag T is now in single-stranded form.
  • An interior (intermediate) portion of the PS is designated CS' .
  • Fig. 4A illustrates a RCC having a TBR near one end and a PS near the other end, with a tag T on the end near PS.
  • PS is hybridized to the end-most portion of TBR, leaving the interior portion IBR
  • 4C CP is shown with a capture segment CS complementary to portion CS' of PS.
  • CP also has a series of attached biotin moieties (shown as B's) .
  • Biotin may be attached enzymatically, chemically, or photochemically.
  • Fig. 4D the hybrid between the opened reagent complex of Fig. 4B and the CP of Fig. 4C is shown.
  • the pendant biotins can now bind to immobilized avidin (shown as Av on support IM) .
  • segment CS• is unavailable in the intact RCC for hybridization to CP. Accordingly, CP can efficiently separate RCCs which have been hybridized to sample polynucleotides G having TNS from those reagent complexes which have not been contacted by TNS.
  • Tag T attached to the solid phase can now be determined.
  • tag T may be specifically released into a fresh solution phase in a variety of ways: (1) displacement with dissolved biotin, (2) melting the double-stranded segment CS/CS' if it is sufficiently short, (3) displacement with either (a) a strand complementary to more of PS than is CS' or (b) a strand complementary to more of CP than is CS or (4) specific chemical cleavage of the link between tag T and segment PS. Modes of release are preferred which do not operate on reagent complexes non-specifically bound to support phase IM. Thus, in many cases, displacement modes (1) and (3) are preferred.
  • Figs. 4A-4D The scheme shown in Figs. 4A-4D is available whether CP was present during hybridization of G to IBR or not. Nevertheless., it is not the only mechanism available. especially when RCC, CP and sample are all admixed together. It should be apparent from Figs. 4A, 4B and 4C that the target can, and usually does, contain a segment identical to segment CS 1 and thus complementary to segment CS, or functionally identical in ability to form a stable hybrid with segment CS under the conditions employed. Thus, especially where CP is provided in excess relative to RCC (each is generally in excess relative to target) , CP may bind to part of TNS, forming a first intermediate structure shown in Fig.. 2E. Most of TNS remains single-stranded in this structure, including a portion ("IBR" 1 ) of TNS which is complementary to IBR or TBR.
  • IBR IBR
  • the first intermediate shown in Fig. 4E, may still bind to the RCC shown in Fig. 4A, with IBR' binding to IBR, forming a second intermediate structure. Strand migration at this point will normally lead to formation of a completely double-stranded TNS/TBR segment. Thus, the structure of Fig. 4B should form with both PS displaced from TBR and CS displaced from the CS' portion of TNS. The same CP, or a different such molecule, may now bind to portion CS' of PS. If, however, the CP/TNS/RCC second intermediate formed when IBR' binds to IBR is stable (strand migration does not displace CP and PS) , then such second intermediate may be a second source of signal specifically bound to support IM.
  • first and second intermediates may, however, be desirable to avoid the formation of first and second intermediates or allow such second intermediates ample time to resolve by branch migration before the original reaction mixture (liquid phase) is removed from support IM. Any such intermediates present at the time when the original reaction mixture is removed from support IM may subsequently complete strand migration and thus detach the signal, unless the particular displaced CP molecule (or an adjacent one on the solid phase also bound to avidin) can find the simultaneously displaced PS segment.
  • Such a second intermediate in a reagent complex having distinct probe strands and label strands may resolve in such fashion to a major extent under certain conditions.
  • the lengths of TBR, IBR, PS and heterologous portions of the probe and of the label strand can, in general, be any of the suitable or preferred lengths indicated in EPA167,238 and 164,876.
  • preferred ranges include about 50-1000 nucleotides for IBR, 20-1000 nucleotides for PS (equal in size to LB in the Figs, of these applications) 70-2000 nucleotides for TBR and 0-15 nucleotides of PS binding to the probe outside of TBR.
  • the portion CS' of PS to which CP can specifically bind once PS is displaced from TBR can be all of PS or most of PS or at one end of PS. It is preferred, however, that each end of CS' be located at least 25, and preferably at least 50 nucleotides from each end of PS (that is, be an intermediate segment of PS) . Such an intermediate segment has reduced susceptibility to becoming momentarily single- stranded under circumstances (sometimes called "breathing") in which the PS/TBR duplex reversibly unwinds (only to subsequently rewind and thus not separate) .
  • PS which have CS f segments that include nucleotides at an end of PS may be captured by CP even without displacement by TNS having occurred. Such non-specific capture could result in non ⁇ specific signal detection unless the rewinding of TBR and PS was effective to displace such CPs which had been non- specifically bound to PS.
  • the length of segment CS' within segment PS is preferably at least 25 nucleotides and more preferably 50-500 nucleotides. At the lower end of the more preferred range for CS ' (50 nucleotides) , it is preferred that PS be at least 100 nucleotide in length (to permit 25 nucleotides on each side of CS 1 ) ' and more preferred that PS be at least 150 nucleotides in length (to permit 50 nucleotides on each side of CS•) .
  • Random introduction of breaks ("nicks") into the normally intact label strand either prior to or during the capture event, can be a source of non-specific signal.
  • Such non-specific signal can also be reduced by the portion of the PS complementary to the CP being internally located within the PS.
  • the preferred segment lengths and positions of the PS and CS described above reduce or eliminate both non-specific capture due to breathing and non-specific signal detection due to such nicking.
  • PS segments binding to portions of the same TBR and multiple TBR regions of the probe, with a label strand having a separate (and different) PS bound to each are also embodiments of this invention.
  • the CP need bind specifically only to the PS of a label strand which had been displaced by sample TNS when that displaced PS has been separated from the immobilized or immobilizable probe.
  • Embodiments are also contemplatedusingmultiplereagent complexes to simultaneously contact a sample to assay for multiple TNS.
  • one or more CPs can be used to separately isolate each group of displaced label strands for separate detection of tags, indicative of the presence and amount of each target.
  • Such separate reagent complexes can be replaced by a probe having multiple label strands attached, each to a portion of a specific TBR. Additionally, the several label strands can be captured simultaneously, but then released for detection sequentially.
  • mismatches may be present is where the binding of TNS to TBR is perfect and the binding of CS to CS' (a portion of PS) is perfect, but the binding of PS to PS' (a portion of TBR) contains mismatches.
  • Such example may prevent rehybridization of CS* to TBR leading to reversal of displacement and loss of signal.
  • This approach may be especially useful for ⁇ ovalent reagent complexes where following displacement the displaced pairing segment is held in close proximity to the TBR.
  • immobilized or immobilizable CPs may be used for either solution or solid phase displacement assays employing either: (1) covalent reagent complexes, (2) reagent complexes with distinct label strands andprobe or (3) covalent IRCs. While covalent attachment has been used herein as an exemplary means for attaching probes or CPs to solid supports or to attach PS to TBR, other forms of attachment such as stable hydrogen bonding (e.g., complementary base pairing) can be employed.
  • Such other forms of attachment can also be employed for post-capture immobilization of an immobilizable CP (e.g., poly-dC on CP immobilized by oligo- dG-cellulose, or similar immobilization by sequences of CP outside of segment CS binding to complementary sequences on a strand immobilized to a solid support or otherwise separable, e.g., by virtue of size).
  • an immobilizable CP e.g., poly-dC on CP immobilized by oligo- dG-cellulose, or similar immobilization by sequences of CP outside of segment CS binding to complementary sequences on a strand immobilized to a solid support or otherwise separable, e.g., by virtue of size).
  • Thevarious reaction conditions e.g., temperature, salt concentration, presence of agents such as recA protein and co actor or polymers such as PEG can be as described in EPA167,238 and 164,876, with the additional proviso of not destabilizing the CS/CS* pairing, once formed.
  • the number of CPs should exceed the number of expected displaced PS. Accordingly, in the usual case where the number of reagent complexes exceeds the anticipated level of TNS, the number of CPs should also exceed anticipated levels of TNS, preferably by a factor of at least 10, more preferably by a factor of at least 100. Thus with 10 ⁇ expected analyte strands having TNS (whether there are 10 7 , 10 8 , 10 9 or 10 10 reagent complexes employed) , there should be at least 10 6 , preferably at least 10 7 , more preferably at 10 8 and commonly 10 9 -10 10 molecules of CP employed.
  • the molar ratio between reagent complexes and CP used in the same assay method (or present in the same kit) does not have independent significance, but rather varies within the range 10 4 :1 to 1:10 4 reagent complexes to CP or even outside of such broad range.
  • the minimum number of CP that may be present is determined by the minimum number required to hybridize a sufficient fraction of displaced label strands in order to result in a signal of the desired intensity within the desired period of time.
  • IRCs were prepared from a nucleic acid construct as follows: Two E. coli plasmids, pMLC12 and pMLC13, each containing ah M13 origin of replication, were prepared by partial Hindlll digestion and Klenow end fill of plasmids pSDL12 and pSDI.13 [A. Levinson et al., J. Mol. Appl. Genet.. 2.: 507-517 (1984)]. pMLC12 and pMLC13 were digested to completion with EcoRI.
  • the large fragment from pMLC12 and the small fragment from pMLC13 were combined, ligated and transformed into MC1061 (F-;hsdR, delta ara-len 7697, araD139, delta lac X 74, galU, galK, rpsL (str r ) ) [See, Y. Casadaban, J. Mol . Biol .. 138 : 179-207 (1980)].
  • Chloramphenical resistant colonies were picked and the correct plasmid, designated pMLC12/13 delta, was identified on the basis of loss of the BamHI cleavage site seen in both pMLC12 and pMLC13.
  • pMLC12/13delta was partially digested with EcoRI so that only one of the two EcoRI sites is digested in most molecules.
  • the partially cut, linearized pMLC12/13delta was isolated following gel electrophoresis.
  • Mp7 DNA was digested with PvuII and a 383 bp fragment was isolated following gel electrophoresis.
  • the PvuII fragment was digested with EcoRI to produce the 52 bp EcoRI fragment containing the Mp7 polylinker and two other PvuII/EcoRI fragments (about 123 bp and 208 bp) .
  • the EcoRI digested PvuII fragment from Mp7 and the linearized, partially EcoRI digested pMLC12/13 elta were ligated, transformed into MC 1061 and chloramphenicol resistant cells were selected. Individual colonies were then grown and DNA was prepared. Plasmids which had correctly incorporated the EcoRI polylinker from Mp7 were identified by the acquisition of a BamHI site and designated pMLC12/13deltaM7. pMLC12 was digested to completion with Hindi.
  • the plasmid pAlbB6 which contains a portion of a human albumin cDNA clone [Lawn et al. , Nucleic Acids Res. , 9_:6103-6114 31
  • F31a was digested to completion with EcoRI and BamHI and the two large fragments were isolated.
  • F31c was digested to completion with EcoRI and Bglll and the small (approximately 550 bp) fragment (between the EcoRI and Bglll sites) was gel isolated.
  • the gel isolated fragments from F31a and F31c were ligated together and transformed into DH1 (ATCC #33849) a recA" bacterium.
  • the recA ⁇ host was used to reduce the possibility of deletion of one or both copies of the inverted repeat through a recA mediated mechanism.
  • Pstl, EcoRI plus Hindlll, or Bglll plus Bam HI was used for further analysis.
  • F41a was digested with EcoRI and Hindlll and blunted with the Klenow fragment of DNA Polymerase I. The approximately 1500 bp fragment was isolated following gel electrophoresis.
  • pMLC12/13deltaM7 was digestedto completion with Ace I and blunted with the Klenow fragment of DNA Polymerase I.
  • the pMLC12/13deltaM7/Acd and Klenow fragment and * the gel-isolated fragment from F41a were joined by DNA ligase and transformed into MC1061. Plasmids which had incorporated the gel isolated fragment were identified by hybridization to the albumin 32 mer and were verified by digestion with Pstl, BamHI, or Xbal. This plasmid was designated pMLC12/13deltaM71VR.
  • Plasmid MpTL poly was prepared by phosphorylating the lower oligonucleotide strand of:
  • Plasmid MpTL poly was confirmed upon sequencing.
  • pMLC12/13deltaM71FR was partially digested with Xbal (which cuts four times within the plasmid) , blunted as above, and full length linearized plasmid DNA was isolated by gel electrophoresis.
  • the plasmid MpTL poly was digested to completion with BamHI and Hindlll and was blunted as above. The 80 bp blunted fragment was isolated, linearized, Xbal partially cut, blunted pMLC12/13deltaM71VR.
  • the DNA was transformed into MC1061 and screened with the oligonucleotide which is complimentary to the TL polylinker. Positive colonies were picked and plasmid which had incorporated the TL polylinker at the correct Xbal site was identified by digestion with Stul and Bgl II and designed pMLC12/13 delta M7IVRTL. pMLC12/13deltaM7IVRTL thus contains fragments from the human albumin gene cloned as inverted repeats in an M13 origin plasmid. Single stranded forms of the construct fold up into a stem loop structure. Cleavage of the mp7 polylinker with a restriction enzyme releases the stem-loop structure from the single-stranded vector backbone.
  • This insert can be used directly as an approximately 1.6 kb nucleotide covalent displacement complex with an approxi ⁇ mately 0.5kb Bgl II-Hinc II albumin fragment as the signal strand, and an approximately 1 kb Bgl II-Pvu II albumin fragment nucleotide TBR.
  • the TBR is located 22 nucleotides from the 5* end and the label strand 41 nucleotides from the 3* end.
  • pMLC12/l ' 3deltaM7IVRTL contains an additional inverted repeat ("IVRTL") located at the inside edge of the label strand, which forms a 52 nucleotide (26 base pair) hairpin containing a double-stranded Eco Rl cleavage site in single stranded forms of this construct. Cleavage at both the Bam HI site in the mp7 polylinker and at the IVRTL Eco Rl site results in the formation of a displacement complex in which the label strand and target strand are held together only by base pairing.
  • IVRTL additional inverted repeat
  • p66b was constructed by gel-isolating the double- stranded PvuII fragment containing the sequence for the entire displacement complex from pMLC12/13deltaM7IVRTL and ligating it to the gel-isolated PvuII backbone of the M13 origin plasmid pUC119. Single-stranded forms of the construct were produced as previously described for pMLC12/13deltaM7IVRTL, except that the DNA was transformed into the E. coli host strain MV1193 [obtained from Dr.
  • Model target was constructed by gel purifying a 2 kb Hindll-Eco Rl fragment from a plasmid pAllAlb which contains the entire cDNA sequence of human albumin.
  • the Hindlll site is the Hindlll site in the 3 ' end of the albumin cDNA.
  • the EcoRI site is present in adjacent vector sequences.
  • the Hindlll-EcoRI fragment was ligated into Hind III-EcoRI digested M13mp8 to give mp8AHAlb. Single stranded DNA was purified from phage containing this construct, and was partially digested with Haelll to linearize these targets. There are no Haelll sites within the albumin cDNA sequence.
  • mpSAllAlb template DNA is complementary to the TBR of p66b displacement complexes.
  • Capture strand mpl9AlbTaqPst was constructed by ligating a 280bp Pstl-Taql segment isolated from a 350bp Bglll-Pstl fragment of human albumin cDNA into Accl/Pstl digested mpl9RfDNA.
  • mp7deltaAlbXbal+ was made by digesting mpl9AlbTaqPst Rf DNA with Xba I, and end filling and gel purifying the resulting 300bp fragment, and ligating it to the 680bp gel purified PvuII vector backbone fragment of mp7.
  • mp7deltaAlb Xbal+ differs from mpl9AlbTaqPst in that a portion of the lac gene and all polylinker cloning sequences are deleted from the mp7delta backbone. Further, the albumin insert in mp7delta AlbXbal+ is complementary to a more interior portion of the pairing segment.
  • Capture strand mp7deltaAlbXbal+ was biotinylated using Photoprobe Biotin (Vector Laboratories) essentially as described by the manufacturer.
  • oligonucleotide was purified away from unincorporated 32P -rATP by centrifuging the reaction twice at 6000 rpm for 30 minutes in a total volume of 500 ul of TE in a Centricon 10 filtration device from Amicon. 10pm kinased oligonucleotide and 2.4pm of Bam HI cut p66b gel purified complex were precipitated together with ethanol, and resuspended in 14 ul of TE.
  • Capturing of displaced label strands can also be accomplished when the CP is immobilized, e.g. , by attachment to latex particles by conventional means.
  • EXAMPLE 3 Capturing and trapping of displaced complex withbiotinylated capture strand p66b Bam complex was labeled at the 3' end by ligation of a 32P-labeled oligonucleotide and purified as described in Example 2. Biotinylated mp7deltaAlbXbal+ was used as the capture strand. Four reactions were set up which included combinations of 0.03pm complex with the following combinations of capture and the Haelll cut mp ⁇ .AllAlb target: Reaction 1 with 0.01 pm target and 0.08 pm capturer; reaction 2 with 0.08 pm capturer only; reaction 3 with 0.01 pm target only; and reaction 4 with neither target nor capturer. All reactions were performed in 50 ul of hybridization buffer, and incubated at 65°C for 60 minutes.
  • the pellet was washed three more times with 500 ul binding buffer and all supernatants and the pellet were counted.
  • the first rinse was then rebound to a fresh 100 ul packed volume aliquot of streptavidin agarose (prewashed with binding buffer as before) in a 5 ml Sarstedt centrifuge tube which was rotated end over end for 15 minutes during binding.
  • the agarose was pelleted by centrifugation, and the supernatant saved and counted.
  • the agarose was washed with 500 ul binding buffer for 15 minutes as above, followed by centrifugation and counting of this supernatant and pellet. Substantially maximal trapping occurred after 15 minutes of incubation.
  • reaction 1 had a % binding of 35%; reaction 2, 3.0%; reaction 3, 1.4% and reaction 4, 1.5%.
  • This experiment demonstrates the use of a biotinylated capturer with a streptavidin agarose trap to collect the captured molecules. Only the reaction which included all three components showed a significant amount (35% of total counts) of label bound to the support.
  • EXAMPLE 4 Large scale displacement and capture with trapping on streptavidin agarose.
  • the Bam p66b covalent displacement complex was labeled to a specific activity of about 10 6 cpm/pm by ligating a 32P-kinased oligonucleotide to the 5' end of the complex with the use of a 21 base splint. 10 pm of the kinased l ⁇ mer (indicated below by the asterisk) , 10 pm of splint, and 1 pm of p66b Bam (underlined below) were incubated together at 22 ⁇ C for 15 minutes in 10 ul of IX ligase buffer. 1 ul of ligase was added and the reaction incubated for an additional 30 minutes. The three molecules form the joint diagrammed below.
  • 25 ul of each reaction was then analyzed by gel electrophoresis and 25 ul by binding to streptavidin agarose as follows. 100 ul packed volume of streptavidin agarose was washed twice in 500 ul binding buffer in a 5 ml Sarstedt tube rotated end over end for 15 minutes, and pelleted by centrifugation. The 25 ul reaction aliquots were diluted to a total of 500 ul binding buffer, and incubated, rotating as above, for 15 minutes. The sample was transferred to an Eppendorf tube for centrifugation and the supernatant was saved.
  • the pellet was rewashed as above, once at room temperature for 15 minutes, then twice at 65"C for 15 minutes, then for 60 minutes at room temperature and finally for 15 minutes at room temperature with lOmM Tris HCl pH8 ImM EDTA [TE] .
  • the final pellet and all supernatants were counted.
  • the percent cpm bound to agarose for each reaction are 1.7% (Reaction 1); 2.5% (Reaction 2); 26.3% (Reaction 3); and 8.7% (Reaction 4).
  • EXAMPLE 5 Prehybridization ofcomplex and target, followedbycapturing Two additional reactions were done using the Bam p66b complex described in Example 4. In these reactions, 0.1 pm complex alone (reaction 1) or 0.1 pm complex and 0.05 pm Haelll cut mp ⁇ AllAlb target (reaction 2) were incubated in 50 ul of hybridization buffer for 30 minutes at 65 ⁇ C. 0.16 pm of biotinylated mp7delta.AlbXbal+ was then added to both reactions. Both reactions were then divided and treated as in Example 4, except that all rinses were at room temperature with binding buffer. By gel analysis, less than 0.05% non ⁇ specific capturing, and approximately 40% specific capturing was observed.
  • Covalent p66d displacement complexes were prepared and labeled at the 5' end by ligation of the kinased 16mer using the EF21 splint as described in Example 4.
  • the specific activity of the resulting complexes was about 1 x 10 6 cpm/pm.
  • Non-covalent p66d complexes were produced by complete digestion of approximately 50 ug of single stranded templated DNA with Bam HI and Eco Rl. Complete digestion was ascertained by the appearance of equimolar amounts of three bands, corresponding to vector backbone, target strand, and signal strand, after electrophoresis of a small aliquot of the digest on an alkaline gel. Non- covalent p66d complexes were labeled at the 5' end of the signal strand as described for covalent complexes, by EF21 splint ligation of a kinased 16mer, with a resultant specific activity of 3 x 10 6 cpm/pm.
  • reactions 1 and 2 contained 0.2 pm target and 0.8 pm capturer with 0.2 pm of either covalent or non-covalent complex, respectively; reactions 3 and 4 were identical to 1 and 2 except they contained no capturer] in a total volume of 50 ul of hybridization buffer [See Example 4] and incubated for 60 minutes at 65 using Haelll digested mpll.AllAlb DNA as target and biotinylated mp7deltaAlbXba3- capturer. 10 ul of each reaction were analyzed by gel electrophoresis nd autoradiography, and the remaining 40 ul by binding to streptavidin agarose. 200 ul packed volume streptavidin agarose was used per reaction.
  • Binding and washing was as described in Example 5 below, except that, after binding, the pellet was rinsed 3 times for 30 minutes at room temperature and once for 60 minutes at 65 ⁇ C. The final pellet and all supernatants were counted. The final cpm bound to agarose are 55.6% (reaction 1), 62.1% (reaction 2), 3.2% (reaction 3), 1.0% (reaction 4). These results show that displacement and capturing are approximately equally effective for covalent and non-covalent complexes. Gel analysis of the same reactions, as well as two reactions in which only displacement complex and capturer were included, demonstrated that 100% of the complexes were displaced by target, and when capturer was included, 100% capturing occurred. In the absence of target, no capturing was observed.
  • second intermediates label capture intermediates which contain capturer hybridized to target which in turn is hybridized to the TBR of the displacement complex
  • Biotinylated nucleic acid strands immobilized by the binding of biotin on the nucleic acid to avidin on a support may be released by displacement with free biotin.
  • the fully protected RM16 was prepared on a 1.0 or more commonly a 7.5 umole scale. With the automated procedure, a 0.2 M solution of the phosphoramidite in dry CH 2 C1 2 was loaded on the synthesizer and the program modified for the addition of this reagent after the detritylation of the last base. In the manual procedure, the support was removed from the synthesis column, dried in vacuo and treated as described below.
  • Acid 16mer d[H0 2 C(CH 2 ) X1 OpCGAAGCTTGGATCCGC] was prepared by a combination of automated and manual solid phase methodologies (see, e.g., Oligonucleotide Synthesis: a Practical Approach, Gait, M.J. ed., IRL Press 1984) on either an Applied Biosystems Model 380A or Beckman Instruments System 1 DNA synthesizer.
  • the RM16 primer was annealed to RM11 template DNA and extended in the presence of excess dCTP, " dGTP and dTTP and 25uCi [alpha-32P]-dATP withKlenow fragment of DNApolymerase for 15 minutes. Excess unlabeled dATP was then added and the reaction was incubated for another 15 minutes. After digestion with the restriction enzyme Avail, the 225 base fragment was purified after electrophoresis on an alkaline agarose gel. This fragment is referred to as labeled 225- 45 mer.
  • Biotinylated labeled 225-mer was bound to avidin in solution at room temperature. Buffer BB+ (Example 8) or excess free biotin in BB+ buffer was then added and the samples were incubated at 65 C for various periods of time. At each time point, the samples were rapidly chilled to 4°C. At the completion of the experiment, all samples were electrophoresed on a 5% non-denaturing acrylamide gel. When the bromophenol blue dye is run far enough into gel, the biotinylated labeled 225-mer separates well (approximately 1cm) from the same biotinylated labeled 225-mer bound to avidin.
  • Streptavidin-agarose (BRL) or streptavidin-latex (Pandex Laboratories) were washed 4X with BB+ (0.2M NaCl, 10 mM Tris-HCl, pH 8.0, 0.01% NP-40) and then resuspended in 1 ml of BB- To each sample was added approximately 40,000 cpm of labeled, biotinylated 225-mer as described above. Following binding for 10 minutes at room temperature, the agarose or latex bound DNA was separated from unbound DNA by centifugation and washing in BB+. The washes included 3 room temperature washes and two washes for 10 minutes each at 65°C. Approximately 75% of the counts bound to the support under these conditions.
  • Each avidin support-DNA complex was then aliquoted into 9 equal reactions and either 800 ul BB (without NP40) or 800 ulBB (without NP-40) containing 1 M free biotin were added to each tube. Following mixing, the samples were placed at 65"C for 1, 3, 10, or 30 minutes. At each time point, the sample was removed from the 65 ⁇ C bath, centrifuged immediately to separate the support from the solution and the supernatant was removed. The pellet was washed twice more with 1 ml BB+ and the supernatants were combined. The agarose or latex pellet was then resuspended in 3 ml BB+ and the pellets and combined supernatants were then counted by Cerenkov counting.
  • M13 DNA (1 ug) was annealed to the M13 Hybridization probe primer (New England Biolabs, 50 pmoles) and extended as described above except that the synthesized DNA was not digested with a restriction endonuclease, and was purified by G-50 spin column chromatography. Approximately 80 x 10 6 cpm of labelled DNA were synthesized and used as probe molecules.
  • EXAMPLE 10 Strand displacement, capturing and biotin displacement.
  • the DNA was then digested with Hind III for 2 hours at 37°C to cut out the 300 base primer extended fragment which is complementary to the insert in mpl9.AlbTaqPst.
  • the DNA was denatured by adding 2 ul of 5 M NaOH and incubating it for 10 minutes at 65 ⁇ C.
  • the primer extended fragment was purified after separation by electrophoresis on a 1% alkaline agarose gel with NA45 paper (Schleicher and Schuell) . DNA yield was estimated by comparison of an aliquot of the capturer with standards on an ethidium stained gel.
  • reaction 3 Three reactions were set up containing 0.2 pm non- covalent complex alone (reaction 3), with 0.5 pm capturer (reaction 2) and with capturer and 0.2 pm target (reaction 1) . Reactions 1 and 2 were incubated for 60 minutes in 50 ul of hybridization buffer at 65 ⁇ C (reaction 3 was not 49 incubated) . 5 ul of reactions 1 and 2 were then removed for gel analysis. The rest of reactions 1 and 2, and reaction 3 were then bound as described in Example 6 to approximately 200 ul packed volume streptavidin agarose.
  • the samples were rinsed by adding 1 ml of binding buffer to the pellet in an eppendorf tube, inverting the tube five times, and centifuging it for 3 minutes. Five rinses were done at room temperature, and a final rinse was done at 65 ⁇ C for 30 minutes with no shaking after the initial 5 inversons. All supernatants and the final pellet were counted.
  • the % cpm band in reaction 1 was 43.6%, in reaction 2, 0.9% and in reaction 3, 2.3%.
  • the gentler washing protocol, or the use of the smaller and singly biotinylated capturer promotes more stable binding of the captured complex to the support. By cutting out and counting the appropriate bands from the gel analysis, it appears that approximately 36% of the captured complexes resolve to form capture-signal strand hybrids, while 64% are apparently present as analyte-complex-capturer intermediates under these reaction conditions.
  • reactions 1 to 3 as described above were used.
  • One ml BB containing 0.1% NP-40 (BB+) was added to each pellet at room temperatue, shaken briefly by inversion and centrifuged to separate the phases (RT wash) .
  • One ml BB+, (at 65°C) was then added and the samples were incubated for 5 minutes at 65 ⁇ C.
  • the samples were then centrifuged to separate the phases and washed twice with one ml BB+ at room temperature.
  • the combined supernatant phases were then pooled (65 ⁇ C/5 minutes/-bio) .
  • the -biotin, 65"C wash was repeated once more for sample 3 only.
  • the signal to noise ratio before the biotin displacement (i.e., the ratio of counts in the reaction 1 pellet/reaction 2 pellet) was 48:1.
  • the improvement brought about by the biotin displacement can be measured by the ratio of % counts released by biotin in fraction 1 over the percent counts released by biotin in either reaction 2 or 3.
  • the improvement is 1.55.
  • the improvement is 14 X.
  • the poor improvement seen in the reaction 2 sample is likely due to the fact that the reaction 2 capturer contains a small amount of M13 polylinker sequence which does result in some capturing by the biotinylated capturer in the absence of target. This capturing, though small, would lead to counts released by biotin.
  • the reaction 3 sample (complex only) represents the type of background most likely to be found in an actual displacement measurement and therefore gives a better representation of the background improvement expected.

Abstract

Les procédés d'analyse diagnostique permettent de détecter la présence d'une séquence de nucléotides cibles (TNS) dans un échantillon biologique. Un complexe réactif utilisé dans une première méthode est également décrit et contient une sonde marquée (LP) ayant une région de liaison cible (TBR) et un second polynucléotide (SP) qui se lie à la sonde dans au moins une portion de la région de liaison cible. Le complexe est mis en contact avec l'échantillon (G) et la séquence de nucléotides cible déplace le second polynucléotide sur le complexe. Le complexe intact est ensuite séparé des hybrides sonde cible, et les hybrides sont détectés. Le second procédé consiste à capturer le brin marqué après déplacement par rapport à la sonde qui se lie sur la cible.
EP19870900593 1985-12-17 1986-12-17 Procede d'analyse de polynucleotides base sur le deplacement des brins, et complexe reactif. Withdrawn EP0286642A4 (fr)

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US80997185A 1985-12-17 1985-12-17
US06/809,992 US4752566A (en) 1985-12-17 1985-12-17 Displacement polynucleotide method and reagent complex employing labeled probe polynucleotide
US809971 1985-12-17
US809992 1991-12-18

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US5359100A (en) * 1987-10-15 1994-10-25 Chiron Corporation Bifunctional blocked phosphoramidites useful in making nucleic acid mutimers
US5124246A (en) * 1987-10-15 1992-06-23 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same
EP0703296B1 (fr) * 1988-09-29 1998-07-22 Chiron Corporation Détermination de polynucléotides par substitution d'un brin sur une sonde de capture
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US5324829A (en) * 1988-12-16 1994-06-28 Ortho Diagnostic Systems, Inc. High specific activity nucleic acid probes having target recognition and signal generating moieties
CA2008096A1 (fr) * 1989-01-19 1990-07-19 Samuel Rose Amplification de l'acide nucleique a l'aide d'une amorce unique
CA2009708A1 (fr) * 1989-02-13 1990-08-13 Jane D. Madonna Sonde d'acide nucleique pour la detection des salmonella pathogene pour les humains
CA2049043A1 (fr) * 1989-03-10 1990-09-11 Mark L. Collins Sondes oligonucleotidiques immobilisees, et leur utilisation
WO1991011533A1 (fr) * 1990-01-26 1991-08-08 E.I. Du Pont De Nemours And Company Procede d'isolement de produits d'extension a partir de reactions de polymerase d'amorces d'adn orientees a l'aide d'un brin complementaire
US5387505A (en) * 1990-05-04 1995-02-07 Eastman Kodak Company Preparation and isolation of single-stranded biotinylated nucleic acids by heat avidin-biotin cleavage
US5445933A (en) * 1992-09-15 1995-08-29 Boehringer Mannheim Corporation Strand displacement assay and complex useful therefor
CA2163393C (fr) * 1994-11-30 2003-04-22 Colleen Marie Nycz Amplification et detection d'acides nucleiques mycobacteriens
US5753439A (en) * 1995-05-19 1998-05-19 Trustees Of Boston University Nucleic acid detection methods
US5616465A (en) 1995-08-09 1997-04-01 The Regents Of The University Of California Detection and isolation of nucleic acid sequences using competitive hybridization probes
US6027879A (en) * 1995-08-09 2000-02-22 The Regents Of The University Of California Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe
WO1999024612A2 (fr) 1997-11-06 1999-05-20 Mosaic Technologies Reactions multiples de deplacement polynucleotidique sequentiel pour amplification et traitement de signaux
AU6412799A (en) * 1998-10-05 2000-04-26 Mosaic Technologies Reverse displacement assay for detection of nucleic acid sequences
WO2001006008A2 (fr) * 1999-07-16 2001-01-25 Aclara Biosciences, Inc. Deplacement de brins multiplexes pour determinations d'acide nucleique
US20060292586A1 (en) * 2004-12-17 2006-12-28 Schroth Gary P ID-tag complexes, arrays, and methods of use thereof
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