WO1991014003A2 - Caracterisation et analyse de loci a nombre variable de repliques en tandem polymorphes - Google Patents

Caracterisation et analyse de loci a nombre variable de repliques en tandem polymorphes Download PDF

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Publication number
WO1991014003A2
WO1991014003A2 PCT/US1991/001628 US9101628W WO9114003A2 WO 1991014003 A2 WO1991014003 A2 WO 1991014003A2 US 9101628 W US9101628 W US 9101628W WO 9114003 A2 WO9114003 A2 WO 9114003A2
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vntr
locus
amplification
primers
sequence
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PCT/US1991/001628
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English (en)
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WO1991014003A3 (fr
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Glenn T. Horn
Brenda L. Richards
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Ig Laboratories, Inc.
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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • VNTR variable number of tandem repeats
  • VNTR polymorphisms are currently analyzed as restriction fragment length polymorphisms (RFLPs) via Southern blot transfer and hybridization to radioactive probes.
  • Southern transfer is used to detect many kinds of polymorphisms , it requires large amounts of sample DNA that is able to be digested by restrict ⁇ ion enzymes, and can take up to a week to complete.
  • a VNTR segment designated pYNZ32 human gene mapping (HGM) locus D4S125) was isolated as part of a family of cosmid clones hybridizing to a synthetic oligonucleotide probe from the zeta globin repeat sequence. Nakamura t a1. , Science 2 jj : 1616- 1622 (1987).
  • the pYNZ32 probe was reported to reveal a six-allele VNTR identified by several enzymes, including Taql and Pstl. Nakamura Y. et al. , Nucl_. Acids Res . 16(9):4186 (1988). It was described as having a heterozygosity of 58% with Taql in 94 unrelated Caucasians and was later closely linked to the locus responsible for Huntington's disease, located on the short arm of chromosome 4. Nakamura Y. et al. , Science 235:1616-1622 (1987); and MacDonald, M.E. et al. , J. Clin. Invest. 84:1013-1016 (1989). As such, the pYNZ32 probe has been used in restriction fragment length polymorphism (RFLP) analysis as a linkage marker for Huntington's disease.
  • RFLP restriction fragment length polymorphism
  • the present invention is a method for determining polymorphisms which occur in VNTR loci, such as the YNZ32 and YNZ22 locus. Primers for YNZ32 are described. According to the method, sequences flanking a VNTR locus of interest are determined and used to construct amplification primers which can direct amplification across the VNTR locus. The primers are extended using polymerase chain reaction (PCR) to produce amplification products which can be directly analyzed. Variations in the length of the amplified products are then used to identify sequence polymorphisms. Sequence variations which do not affect the size of the amplification products can be further analyzed by digesting the amplification products with various restriction enzymes to generate restriction fragments . The invention can be used in genetic linkage studies, paternity testing, forensic identification or as a diagnostic for polymorphisms.
  • PCR polymerase chain reaction
  • Figure 1 shows a restriction map of the pYNZ32 insert. Restriction sites are indicated as R, Rsal; P, Pstl; T, Taql; H, Hindlll; B, Bgll; M, Mspl. The hatched portion indicates the repeat region. The double-thick lines represent the areas from which the sequence data in Figure 2 was taken. Arrows beneath the map show approximate positions for the amplifi ⁇ cation primers.
  • Figure 2 shows the partial nucleotide sequence of plasmid pYNZ32.
  • Figure 3 shows sequence diversity within the repeated region of pYNZ32 which is shown in Figure 2.
  • Figure 4A shows the results of agarose gel electrophoresis of PCR products amplified from total genomic DNA.
  • Lanes 1-3 amplification of DNA from a father (showing allelic products designated A and F) , a child (products F and G) , and a mother (C and G) ;
  • lanes 4,5,7,8 amplification of DNA from 4 unrelated indi ⁇ viduals giving amplified products B and E, A and E, D and I, and D and G, respectively;
  • lane 6 Rsal DNA marker fragments from bacteriophage ⁇ . Sizes in bp are indicated to the right.
  • Figure 4B shows a Southern blot analysis of Taql digested DNA of some of the same samples amplified above, and hybridized with probe pYNZ32. Overloading of the first two lanes has slightly reduced the apparent size of the bands. The exposure of these lanes has been reduced photographically. Sizes in Kbp are shown on the right.
  • Figure 4C is a schematic showing the relative positions of the YNZ32 amplified products versus the ⁇ DNA/Rsal marker fragments.
  • the largest marker frag ⁇ ment represents a doublet of 2196 and 2183 bp , and the smallest fragment shown is 1436 bp .
  • Figure 5 shows Mspl restriction maps for twelve YNZ32 amplified allele products.
  • the products are designated A to I (largest to smallest), and some products of the same size are shown which differ in their Mspl digestion pattern.
  • the alleles are aligned at the conserved Mspl site, the second site in most of the alleles (except for I which has only one Mspl site) .
  • a star indicates the presence of the unique Bgll site.
  • This invention pertains to methods for amplifying polymorphic VNTR loci and to methods for determining polymorphisms which occur at the VNTR locus.
  • PCR poly erase chain reaction
  • nucleotide sequences flanking a VNTR locus of interest are first determined, From that information, two oligonucleotide primers are designed to direct amplification across the VNTR region.
  • One oligonucleotide primer comprises a nucleotide sequence which hybridizes to the region flanking one end of the VNTR sequence.
  • the second primer comprises a nucleotide sequence which hybridizes to the region of flanking the other end of the VNTR sequence. Hybridization of the primers to other portions of the VNTR can be prevented by restricting the primers to those nucleotides which are unique to the flanking sequences and avoiding segments of DNA which are partially homologous to the repeat unit.
  • the primers are then subjected to conditions sufficient to direct amplification across the VNTR locus. This can be accomplished using PCR techniques described by Mullis, K.B. et al. , U.S. Patent No. 4,683,195 and Mullis, K.B. , U.S. Patent No. 4,683,202. The teachings of these patents are incorporated by reference herein.
  • the resulting amplification products can then be analyzed for size variations using gel electrophoresis techniques. Variations observed in the length of the fragments are indicative of sequence polymorphisms . Sequence polymorphisms which do not affect the length of the PCR product can be determined by digesting them with various restriction enzymes to produce fragments from which a restriction map can be determined. The lengths of the fragments are indicative of sequence polymorphisms .
  • the methods of this invention provide rapid analysis of polymorphisms at VNTR loci using small amounts of sample DNA. Such analysis of these regions is extremely useful in genetic linkage studies, paternity testing and forensic identification.
  • One advantage is that cumbersome RFLP-based assays are not needed.
  • the present invention is based on the characteri ⁇ zation of the highly polymorphic VNTR locus pYNZ32, which had been previously isolated. Nakamura, Y. et *___• - Science 235:1616-1622 (1987). For the first time, a restriction map for this locus has been determined, as well as the nucleotide sequence of a substantial portion of this locus.
  • oligonucleotides derived from this sequence can be synthesized. Such synthetic oligonucleotides are useful as primers for amplification techniques and can be used to direct amplification across the VNTR region. DNA sequencing in the areas within and flanking the repeated segment allowed for the design of specific amplification primers.
  • the repeated region of pYNZ32 consists of an imperfectly duplicated 27 base pair motif, 16 bases of which are more highly conserved. Allelic products from PCR amplification were resolved into nine different size classes ranging from approxi ⁇ mately 1400 to 2200 base pairs. Additional poly- morphism was revealed when the amplified products were analyzed by restriction enzyme digestion.
  • the cloned insert in probe pYNZ32 was mapped for various restriction sites (Figure 1) .
  • the distance between the two Taql sites suggests they are responsible for the known Taql RFLP (2.3-2.8 Kbp) for YNZ32 on human genomic DNA Southern blots. Nakamura Y. et al. , Science 235:1616-1622 (1987).
  • the Pstl site is most likely involved with the Pstl polymorphism, with the other site lying further to the right of the segment contained within the cloned fragment.
  • Mspl RFLP range of 1.2-1.8 Kbp for YNZ32 on genomic DNA Southern blots (Nakamura Y. e t a ⁇ . , Nucl ⁇ _Acijis_Res ⁇ 16:4186 (1988))
  • four fragments in the 400-700 bp range were observed, consistent with the map of the cloned probe ( Figure 1) .
  • the sequences flanking the repeat region were also characterized. The information was used to construct primers for amplification of the repeat region.
  • Primer GH301 and GH290 were used to amplify the YNZ32 locus from human genomic DNA samples using polymerase chain reaction techniques.
  • the amplifica ⁇ tion products ranged in size from approximately 1400 to 2200 base pairs.
  • These products were grouped into 9 size classes designated A to I in order of decreasing size (amplification from the pYNZ32 clone yielded a "D" allele) , according to their positions relative to the fragments in Rsal digested lambda DNA ( Figures 4A and 4C) .
  • Each size class encompasses a narrow range of amplified allelic products differing slightly in length.
  • allelic size classes indicate that additional polymorphism might be elucidated by restriction enzyme mapping.
  • Several amplified products were therefore isolated, reamplified, and analyzed by digestion with Mspl or Bgll . Twelve of these products having different digestion patterns were also mapped with Mspl ( Figure 5). Most of the products have three Mspl sites; the maps in Figure 5 are lined up at the second Mspl site, which is conserved in all but one of the alleles.
  • Allelic products Al and Fl are missing the first Mspl site, while products Bl, Cl and Gl are missing the third Mspl site.
  • the II allele has only one internal Mspl site.
  • the sequence of the repeated region of pYNZ32 shows several interesting features.
  • the 27 bp repeated motif is neither conserved nor exactly tandemly ar- ranged, in contrast with other VNTR loci. This sug ⁇ gests that YNZ32 may have once been a perfect tandem repeat which has since diverged. This sequence diver ⁇ gence of the repeat may prevent changes in size seen in some other VNTR loci. Jeffreys, A.J. et al. , Nature H2:278-281 (1988). We were unable to detect major similarities between this sequence and the pYNZ22 repeat (Wolff, R.K. et a1.
  • a search of sequences listed in GenBank revealed the greatest similarity to a polypyrimidine repeat isolated from the mouse genome. Deugau, K.V. et 1. , Anal ⁇ _Biochem ⁇ 129:88-97 (1983) .
  • Amplification of the YNZ32 locus by PCR is a rapid alternative to analysis by other assays. Both the amplif cation and agarose gel analysis of YNZ32 can be performed within four hours.
  • the direct visualization of PCR products on ethidium bromide-stained agarose gels allows the allelic products to be better resolved than the bands seen on Southern blots. Amplified products are also amenable to restriction enzyme analysis, which reveals additional polymorphism.
  • the YNZ32 locus contains poly ⁇ morphic restriction sites.
  • This additional poly ⁇ morphism can be used to discriminate alleles of similar size, thereby increasing the diagnostic utility of this locus.
  • the Bl and B2 alleles ( Figure 5) give the same size PCR products, but when digested with Mspl, release distinctive fragments of 775 or 605 bp , respectively. Similar discrimination between certain alleles can also be achieved using the single Bgll site, which is present only in some chromosomes.
  • the clone pYNZ32 was obtained from Y. Nakamura and is also available from the ATCC (#57548). It has a total insert size of 4.1 Kbp.
  • High molecular weight genomic DNA used for amplification was prepared by standard methods (Maniatis T. et al. , "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). Oligonucleotide primers were synthesized on an Applied Biosystems 380B Synthesizer. Agarose gels were made from SEAKEM GTG agarose (American Bioanalytical) and were run in IX TBE buffer with 0.1 ⁇ g/ml ethidium bromide at 10 volts per cm.
  • the pYNZ32 clone was mapped with several re ⁇ striction enzymes. The positions of Mspl sites were more accurately determined by partial Mspl digestion of
  • DNA fragments flanking and within the repeated region of pYNZ32 were subcloned and sequenced ( Figure 2).
  • the first segment shown here runs from the left Taql site until about 130 base pairs into the repeat. Also shown are the locations of the Mspl and Pstl restriction sites used in RFLP analysis. The arrow indicates the location and direction of the GH301 oligonucleotide primer used in PCR amplification. The repeated region begins just after the Pstl site, and the more highly conserved core segment (Figure 3) is underlined.
  • the second segment shows the final 130 base pairs of the repeat, the location and orientation of the GH290 primer, and the sequence flanking the repeated region. Additional sequence was determined between these segments and is include in Figure 3, but could not be accurately placed due to the repetitive nature of this region.
  • PCR amplification reactions were performed using AmpliTaq DNA polymerase and a DNA Thermal Cycler (Perkin Elmer) .
  • One ⁇ g of human genomic DNA was amplified for 28 cycles of 10 seconds at 94°C, 10 seconds at 55 ⁇ C, and 30 seconds at 74 ⁇ C, with a final soak for 10 minutes at 74°C.
  • the reaction buffer used was described previously (Saiki, R.K. et al.. , ⁇ £i. nc:e 129:487-491 (1988)), with the exception that KC1 was not included, and the concentration of MgCl- was reduced to about 1.2mM.
  • the amplification should be optimized empirically.
  • the yield of PCR product was also reduced when amplification primers were used at less than I ⁇ H .
  • PCR products were analyzed on 1% agarose gels.
  • a Rsal digest of bacteriophage ⁇ DNA was used as markers for the amplified alleles since it has several fragments in the appropriate size range.
  • Amplified allelic products are designated A to I (largest to smallest) by their positions relative to these size markers ( Figure 4C) . Those products within a single size class but having internal variation are distinguished by numbers (e.g. , product Bl vs. B2) . Further analysis of the amplified DNA was performed by electroeluting the DNA from a gel slice (Maniatis, T. et al.

Abstract

Caractérisation du locus à nombre variable de répliques en tandem (VNTR) YNZ32 et procédés de détection de polymorphismes au niveau d'un locus VNTR hautement polymorphe. Les polymorphismes sont détectés par amplification du locus VNTR et par digestion enzymatique des produits d'amplification. Les variations observées dans les cartes de restriction des fragments sont ensuite utilisées afin d'évaluer les polymorphismes. L'invention concerne également des amorces permettant l'amplification VNTR du locus YNZ32.
PCT/US1991/001628 1990-03-09 1991-03-11 Caracterisation et analyse de loci a nombre variable de repliques en tandem polymorphes WO1991014003A2 (fr)

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0534858A1 (fr) * 1991-09-24 1993-03-31 Keygene N.V. Amplification sélective des fragments de restriction: procédé général pour le "fingerprinting" d'ADN
WO1993011264A1 (fr) * 1991-12-04 1993-06-10 E.I. Du Pont De Nemours And Company Procede d'identification de micro-organismes par amplification d'adn arbitraire et dirigee
US5919626A (en) * 1997-06-06 1999-07-06 Orchid Bio Computer, Inc. Attachment of unmodified nucleic acids to silanized solid phase surfaces
US6808633B1 (en) 1999-06-23 2004-10-26 Hitachi, Ltd. Centrifugal separator and sample preparation device using the separator
US7026115B1 (en) 1991-09-24 2006-04-11 Keygene N.V. Selective restriction fragment amplification: fingerprinting
US7329490B2 (en) 2003-09-15 2008-02-12 Bristol-Myers Squibb Company Methods for diagnosing schizophrenia by detecting a polymorphism in the KalphaM1 gene
EP1950305A1 (fr) 2001-05-09 2008-07-30 Monsanto Technology, LLC Gènes tyr et utilisations associées
EP2080766A1 (fr) 2001-06-06 2009-07-22 Bristol-Myers Squibb Company Acides nucléiques et polypeptides apparentés à B7 utiles pour l'immunomodulation
US7935488B2 (en) 1991-09-24 2011-05-03 Keygene N.V. Selective restriction fragment amplification: fingerprinting
WO2012104851A1 (fr) 2011-01-31 2012-08-09 Yeda Research And Development Co. Ltd. Méthodes de diagnostic d'une maladie utilisant une pcr d'extension-chevauchement
US8455190B2 (en) 2007-08-01 2013-06-04 Dana-Farber Cancer Institute, Inc. Enrichment of a target sequence
US9133490B2 (en) 2012-05-16 2015-09-15 Transgenomic, Inc. Step-up method for COLD-PCR enrichment
US9957556B2 (en) 2010-03-08 2018-05-01 Dana-Farber Cancer Institute, Inc. Full COLD-PCR enrichment with reference blocking sequence
US10913977B2 (en) 2013-07-24 2021-02-09 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable enrichment of minor DNA alleles by limiting denaturation time in PCR or simply enable enrichment of minor DNA alleles by limiting the denaturation time in PCR
US11130992B2 (en) 2011-03-31 2021-09-28 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable multiplex COLD-PCR
US11174511B2 (en) 2017-07-24 2021-11-16 Dana-Farber Cancer Institute, Inc. Methods and compositions for selecting and amplifying DNA targets in a single reaction mixture
US11371090B2 (en) 2016-12-12 2022-06-28 Dana-Farber Cancer Institute, Inc. Compositions and methods for molecular barcoding of DNA molecules prior to mutation enrichment and/or mutation detection

Citations (2)

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Publication number Priority date Publication date Assignee Title
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EP0370719A2 (fr) * 1988-11-25 1990-05-30 Imperial Chemical Industries Plc Des séquences nucléotidiques élongées

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Publication number Priority date Publication date Assignee Title
WO1989007658A1 (fr) * 1988-02-18 1989-08-24 University Of Utah Identification genetique avec des adn d'investigation ayant des sites repetitifs en tandem en quantite variable
EP0370719A2 (fr) * 1988-11-25 1990-05-30 Imperial Chemical Industries Plc Des séquences nucléotidiques élongées

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Title
Biological Abstracts, volume 91, no. 8, April 1991, B. Richards et al.: "Characterization and rapid analysis of the highly poly-morphic VNTR locus D4S125 (YNZ32), closely linked to the huntington disease gene", see page 461 *
Nucleic Acids Research, volume 17, no. 5, March 1989, IRL Press, G.T. Horn et al.: "Amplification of a highly polymorphic VNTR segment by the polymerase chain reaction", page 2140 *
Science, volume 235, March 1987, Yusuke Nakamura et al.: "Variable number of tandem repeat (VNTR) markers for human gene mapping", pages 1616-1622 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7026115B1 (en) 1991-09-24 2006-04-11 Keygene N.V. Selective restriction fragment amplification: fingerprinting
WO1993006239A1 (fr) * 1991-09-24 1993-04-01 Keygene N.V. Amplification selective de fragments de restriction: procede general de dactyloscopie genetique
US7935488B2 (en) 1991-09-24 2011-05-03 Keygene N.V. Selective restriction fragment amplification: fingerprinting
EP0969102A2 (fr) * 1991-09-24 2000-01-05 Keygene N.V. Amplification sélective des fragments de restriction; procédé général pour le "fingerprinting" d'ADN
EP0969102A3 (fr) * 1991-09-24 2000-02-23 Keygene N.V. Amorcées, kits et fragments de restriction et son utilisation dans l'amplification sélective des fragments de restriction
US6045994A (en) * 1991-09-24 2000-04-04 Keygene N.V. Selective restriction fragment amplification: fingerprinting
US7572583B2 (en) 1991-09-24 2009-08-11 Keygene N.V. Selective restriction fragment amplification: fingerprinting
EP0534858A1 (fr) * 1991-09-24 1993-03-31 Keygene N.V. Amplification sélective des fragments de restriction: procédé général pour le "fingerprinting" d'ADN
WO1993011264A1 (fr) * 1991-12-04 1993-06-10 E.I. Du Pont De Nemours And Company Procede d'identification de micro-organismes par amplification d'adn arbitraire et dirigee
US5753467A (en) * 1991-12-04 1998-05-19 E. I. Du Pont De Nemours And Company Method for the identification of microorganisms by the utilization of directed and arbitrary DNA amplification
US5919626A (en) * 1997-06-06 1999-07-06 Orchid Bio Computer, Inc. Attachment of unmodified nucleic acids to silanized solid phase surfaces
US6387626B1 (en) 1997-06-06 2002-05-14 Orchid Biosciences, Inc. Covalent attachment of unmodified nucleic acids to silanized solid phase surfaces
US6136962A (en) * 1997-06-06 2000-10-24 Orchid Biosciences, Inc. Covalent attachment of unmodified nucleic acids to silanized solid phase surfaces
US6808633B1 (en) 1999-06-23 2004-10-26 Hitachi, Ltd. Centrifugal separator and sample preparation device using the separator
EP1950305A1 (fr) 2001-05-09 2008-07-30 Monsanto Technology, LLC Gènes tyr et utilisations associées
EP2080766A1 (fr) 2001-06-06 2009-07-22 Bristol-Myers Squibb Company Acides nucléiques et polypeptides apparentés à B7 utiles pour l'immunomodulation
US7329490B2 (en) 2003-09-15 2008-02-12 Bristol-Myers Squibb Company Methods for diagnosing schizophrenia by detecting a polymorphism in the KalphaM1 gene
US8455190B2 (en) 2007-08-01 2013-06-04 Dana-Farber Cancer Institute, Inc. Enrichment of a target sequence
US9957556B2 (en) 2010-03-08 2018-05-01 Dana-Farber Cancer Institute, Inc. Full COLD-PCR enrichment with reference blocking sequence
US11174510B2 (en) 2010-03-08 2021-11-16 Dana-Farber Cancer Institute, Inc. Full COLD-PCR enrichment with reference blocking sequence
WO2012104851A1 (fr) 2011-01-31 2012-08-09 Yeda Research And Development Co. Ltd. Méthodes de diagnostic d'une maladie utilisant une pcr d'extension-chevauchement
US11130992B2 (en) 2011-03-31 2021-09-28 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable multiplex COLD-PCR
US9133490B2 (en) 2012-05-16 2015-09-15 Transgenomic, Inc. Step-up method for COLD-PCR enrichment
US10913977B2 (en) 2013-07-24 2021-02-09 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable enrichment of minor DNA alleles by limiting denaturation time in PCR or simply enable enrichment of minor DNA alleles by limiting the denaturation time in PCR
US11371090B2 (en) 2016-12-12 2022-06-28 Dana-Farber Cancer Institute, Inc. Compositions and methods for molecular barcoding of DNA molecules prior to mutation enrichment and/or mutation detection
US11174511B2 (en) 2017-07-24 2021-11-16 Dana-Farber Cancer Institute, Inc. Methods and compositions for selecting and amplifying DNA targets in a single reaction mixture

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