WO2002024960A1 - Detection de la variation d'adn - Google Patents

Detection de la variation d'adn Download PDF

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Publication number
WO2002024960A1
WO2002024960A1 PCT/US2001/029922 US0129922W WO0224960A1 WO 2002024960 A1 WO2002024960 A1 WO 2002024960A1 US 0129922 W US0129922 W US 0129922W WO 0224960 A1 WO0224960 A1 WO 0224960A1
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WIPO (PCT)
Prior art keywords
dna
nucleic acid
version
probes
duplex
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PCT/US2001/029922
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English (en)
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Qinghong Yang
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Freshgene Inc
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Freshgene Inc
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Priority to AU2001294687A priority Critical patent/AU2001294687A1/en
Publication of WO2002024960A1 publication Critical patent/WO2002024960A1/fr
Anticipated expiration legal-status Critical
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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
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to the field of molecular biology, more particularly nucleic acid hybridization, Holiday junction formation and branch migration.
  • the invention provides methods and reagents for detecting the presence of a difference between two related nucleic acid sequences.
  • the difference is a mutation, such as a point mutation, deletion or insertion.
  • Practical applications of the invention include, but are not limited to, genotyping, discovery and detection of single nucleotide polymorphisms, characterization and quantitation of polynucleotides, mutation rate detection, gene expression analysis.
  • the invention is capable of distinguishing between homozygous and heterozygous genetic variation
  • nucleic acids to bind selectively and specifically to complementary nucleic acid sequence has been exploited in the development of numerous nucleic acid hybridization techniques. Not only are such techniques useful for detecting complementarity and/or identity between nucleic acid sequences (e.g.: quantitating differential gene expression level such as Northern blots, Southern blots and gene expression chip/micro-arrays), but in some cases they are exploited to be used for detecting differences between related nucleic acid sequences.
  • Affymetrix's SNP-chip and various micro-array-based SNP scoring chip are examples of a current genotyping technology that is based on allele-specific hybridization.
  • the hybridization is stronger between two perfectly complementary DNA strands than that between two non-perfectly complementary DNA strands (including those that have either a single or multiple base-pair-mismatch between the two complementary strands).
  • the single- base-pair difference is usually too small to render a high enough specificity for SNP scoring.
  • the method disclosed in this patent application addresses the problem by combining highly specific allele-specific holiday structure formation with nucleic acid hybridization techniques (e.g.: gene chip/micro-array or fluorescence-labeled beads).
  • nucleic acid hybridization techniques e.g.: gene chip/micro-array or fluorescence-labeled beads.
  • a single-stranded oligo that is completely (or partially) complementary to a specific part of single-stranded M13mpl8 viral DNA anneals to the viral DNA and form a partial duplex with either 1 (or 2) tail(s) at each end.
  • the partial duplex formed between the oligo and the M13mpl8 viral DNA can then form a four-way Holiday-like structure with an invading partial duplex with either 1 (or 2) complementary tails.
  • the four-way Holiday-like structure then undergoes branch migration in the direction away from the tail(s) (It can not branch migrate back towards the tail(s) due to energy barrier: breaking existing H-bonds without forming new ones).
  • a single (or multiple) base pair difference between the duplex part of oligo M13mpl8 partial duplex and the duplex part of the invading partial duplex poses enough energy barrier (2 H-Bonds -> 0 H-bond) to impede the branch-migration and prevent the release of the annealed oligo, regardless of the presence or absence of Mg++.
  • a DNA sample of interest (or target DNA, e.g.: genomic DNA or other DNA preparations that need to be genotyped) are immobilized on a solid surface.
  • the DNA sample can be immobilized on a piece of nitrocellulose paper, baked and followed by UN cross-linking (standard procedure as in Southern blot).
  • the target D ⁇ A can be denatured first and then immobilized on the solid surface, or it can be immobilized on the solid surface first and then denatured.
  • a collection of probes are mixed with the immobilized and denatured target D ⁇ A.
  • the collection of probes is comprised of n (1-10,000,000) different probes, each targeted at a specific S ⁇ P ( Figure 1, only 4 different probes targeted for 4 S ⁇ Ps are shown).
  • Each probe is comprised of three parts (figure 1):
  • Trn (Trl,Tr2,Tr3).
  • Trn 0 bp
  • Trn the partial duplexes formed between the probes and their target D ⁇ A have a single tail at one end.
  • Sequences for Trn are not found in the target D ⁇ A sample and therefore will not hybridize with the target D ⁇ A.
  • sequences for Trn can derive from bacteria specific sequences that have no homology with the human D ⁇ A.
  • sequences for T are not found in the target D ⁇ A sample and therefore will not hybridize with the target D ⁇ A.
  • sequences for Trn can derive from bacteria specific sequences that have no homology with the human D ⁇ A.
  • washing buffer used for standard southern blot can be used.
  • buffer e.g.: many commonly used buffers including TES buffer (50mM-Tris-Hcl(PH 7.5), 50mM NaCl, lmM-EDTA), TSM buffer (50mM-Tris- Hcl(PH 7.5), 25mM NaCl, lOmM MgCl 2 , lmM-EDTA), PCR buffers (with Mg++ for double-tailed partial duplexes and PCR buffers with/without Mg++ for single- tailed partial duplexes)) at certain temperature (10°C - 75°C, preferably, 37°C - 65°C), the reference partial DNA duplexes form holiday structures with their corresponding partial duplexes formed (in step 2) between the immobilized target DNA and corresponding probes.
  • buffer e.g.: many commonly used buffers including TES buffer (50mM-Tris-Hcl(PH 7.5), 50mM NaCl, lmM-EDTA), TSM buffer (50mM
  • the formed Holiday structures will undergo branch migration (1 minute -240 minutes, in certain buffer (e.g.: many commonly used buffers including TES buffer (50mM-Tris-Hcl(PH 7.5), 50mM NaCl, lmM- EDTA), TSM buffer (50mM-Tris-Hcl(PH 7.5), 25mM NaCl, lOmM MgCl 2 , lmM-EDTA), PCR buffers (with Mg++ for double-tailed partial duplexes and PCR buffers with/without Mg++ for single-tailed partial duplexes)) at certain temperature (10°C - 75°C, preferably, 37°C - 65°C)).
  • buffer e.g.: many commonly used buffers including TES buffer (50mM-Tris-Hcl(PH 7.5), 50mM NaCl, lmM- EDTA), TSM buffer (50mM-Tris-Hcl(PH 7.5), 25mM NaCl,
  • a partial duplex formed (in step 2) between the immobilized target DNA and corresponding probes contains a homo-duplex version (e.g: target DNA version 1 anneal with probe DNA version 1, or alternatively, target DNA version 2 anneal with probe version 2) that is different from the homo-duplex version of the reference partial DNA duplex it forms a Holiday junction with, branch migration of that Holiday junction will stop and the probe will not be release from the immobilized target DNA (2 H-bonds-i 0 H-bonds, energy barrier).
  • a homo-duplex version e.g: target DNA version 1 anneal with probe DNA version 1, or alternatively, target DNA version 2 anneal with probe version 2 that is different from the homo-duplex version of the reference partial DNA duplex it forms a Holiday junction with, branch migration of that Holiday junction will stop and the probe will not be release from the immobilized target DNA (2 H-bonds-i 0 H-bonds, energy barrier).
  • a partial duplex formed (in step 2) between the immobilized target DNA and corresponding probes contains a homo-duplex version (e.g: target DNA version 1 anneal with probe DNA version 1, or alternatively, target DNA version 2 anneal with probe version 2) that is the same as the homoduplex version of the reference partial DNA duplex it forms a Holiday junction with, branch migration of that Holiday junction will proceed all the way through and the probe will be release from the immobilized target DNA due to complete strand exchange (2 H-bonds- 2 H-bonds, no energy barrier).
  • a homo-duplex version e.g: target DNA version 1 anneal with probe DNA version 1, or alternatively, target DNA version 2 anneal with probe version 2 that is the same as the homoduplex version of the reference partial DNA duplex it forms a Holiday junction with
  • a partial duplex formed (in step 2) between the immobilized target DNA and corresponding probes contains a hetero-duplex version (e.g: target DNA version 1 anneal with probe DNA version 2, or alternatively, target DNA version 2 anneal with probe version 1)
  • the Holiday junction it form with either of the two homo-duplex versions of the reference partial DNA duplex will be resolved due to complete branch migration and the probe will be release from the immobilized target DNA due to complete strand exchange (1 H-bond-M H- bond, no energy barrier).
  • a partial duplex formed (in step 2) between the immobilized target DNA and corresponding probes contains a hetero-duplex version (e.g: target DNA version 1 anneal with probe DNA version 2, or alternatively, target DNA version 2 anneal with probe version 1)
  • the Holiday junction it form with either of the two hetero-duplex versions of the reference partial DNA duplex will be resolved due to complete branch migration and the probe will be release from the immobilized target DNA due to complete strand exchange (either 2 H- bonds- 2 H-bonds or 0 H-bond -> 2 H-bonds, both cases no energy barrier).
  • Each reference partial duplex is comprised of (figure 1, page 1) two strands:
  • First strand is completely complementary to the target DNA at a specific SNP position.
  • This strand is comprised of (one version of, same or different) the middle part of its corresponding probe in step 2 plus the sequences flanking that middle part at both the left and the right side ( Figure l, page 1).
  • the other strand is comprised of 3 parts (Figure 1, page 1): a. A middle part that is perfectly complementary to the middle part of the first strand. b. A 0-80 bp long (preferably Obp or 10-50 bp) 5' tail Un (U1,U2,U3...)- Sequences for Trn are not found in the target DNA sample and therefore will not hybridize with the target DNA. For example, for human genotyping, sequences for Trn can derive from bacteria specific sequences that have no homology with the human DNA. c.
  • Trn' (Trl',Tr2',Tr3 ⁇ ..) 3' tail Trn' (Trl',Tr2',Tr3 ⁇ ..) that is complementary to and can anneal with tail Trn in the corresponding probe.
  • Trn' 0 bp
  • the reference partial duplexes have a single tail at one end.
  • Sequences for Trn' are not found in the target DNA sample and therefore will not hybridize with the target DNA.
  • sequences for Trn' can derive from bacteria specific sequences that have no homology with the human DNA.
  • the released probes selectively amplified are hybridized with DNA chip/micro-array or (fluorescent) beads that are immobilized/coated with the DNA sequences between T' and UR for each of the n SNPs of interest.
  • the presence or absence of the released oligos for a specific SNP can be detected by monitoring the amplification of the released oligos (e,g,: using fluorescent dyes such as PicoGreen or Ethidium Bromide).
  • the probes can be labeled, for an example, with magnet beads and the released probes due to complete strand exchange can then be separated from reference DNA and isolated via magnet.
  • the identification of the isolated released probes can be obtained by using hybridization with DNA chip/micro-array or (fluorescent) beads that are immobilized/coated with the n SNPs of interest.
  • Tabel 1 shows different bar code for three different genotypes at a specific SNP position by using the above scoring method.
  • SNPs with highly accurate but expensive genotyping assays for the target DNA and then use these SNPs as external control.
  • b Mix some control DNA (with known sequences that are not found in target DNA) at comparable concentration with the target DNA before immobilization. Add control probes at comparable concentration in the probe pool before hybridization with the immobilized DNA. Also add control reference partial DNA duplexes in the reference partial DNA duplex pool. Correct or wrong scoring of these control DNA can give you a good estimation about the quality of each assay performed.
  • the probes released from version 1 probe with version 1 reference partial DNA duplex are labeled with both label X (e.g. red label) and label Y (e.g.: green label), one label at a time.
  • the probes released from version 1 probe with version 2 reference partial DNA duplex are also labeled with both label X (e.g.: red label) and label Y (e.g. green label), one at a time.
  • the X-labeled/amplified pool of probes released from version 1 probe with version 1 reference partial DNA duplex are mixed with the Y-labeled/amplified pool of probes released from version 1 probe with version 2 reference partial DNA duplex before hybridization with the chip/micro-array.
  • the Y- labeled/amplified pool of probes released from version 1 probe with version 1 reference partial DNA duplex are mixed with the X- labeled/amplified pool of probes released from version 1 probe with version 2 reference partial DNA duplex before hybridization with the chip/micro-array.
  • the ratio the intensity of red signal vs. green signal will be scored instead of the absolute intensity of red or green signal.
  • many different schemes of scoring can be apparent to a skilled researcher in molecular biology. eve optimal accuracy, the internal controls and external controls can be combined to produce many different schemes for SNP scoring based on the method disclosed here.

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Abstract

La présente invention concerne des procédés et des réactifs qui permettent de détecter la présence d'une différence entre deux séquences d'acides nucléiques proches. Dans certaines formes de réalisation de l'invention, la différence est une mutation, telle qu'une mutation ponctuelle, une suppression ou une insertion.
PCT/US2001/029922 2000-09-22 2001-09-24 Detection de la variation d'adn Ceased WO2002024960A1 (fr)

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AU2001294687A AU2001294687A1 (en) 2000-09-22 2001-09-24 Detection of dna variation

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US23485700P 2000-09-22 2000-09-22
US60/234,857 2000-09-22

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013439A (en) * 1995-12-22 2000-01-11 Dade Behring Marburg Gmbh Detection of differences in nucleic acids
WO2000020643A1 (fr) * 1998-10-05 2000-04-13 Mosaic Technologies Analyse par deplacement inverse pour la detection de sequences d'acide nucleique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013439A (en) * 1995-12-22 2000-01-11 Dade Behring Marburg Gmbh Detection of differences in nucleic acids
WO2000020643A1 (fr) * 1998-10-05 2000-04-13 Mosaic Technologies Analyse par deplacement inverse pour la detection de sequences d'acide nucleique

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