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WO1999023257A1 - Procede de detection d'adn mutant par mipc et pcr - Google Patents

Procede de detection d'adn mutant par mipc et pcr Download PDF

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Publication number
WO1999023257A1
WO1999023257A1 PCT/US1998/023265 US9823265W WO9923257A1 WO 1999023257 A1 WO1999023257 A1 WO 1999023257A1 US 9823265 W US9823265 W US 9823265W WO 9923257 A1 WO9923257 A1 WO 9923257A1
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WO
WIPO (PCT)
Prior art keywords
dna
sample
heteroduplex
wild type
mutant
Prior art date
Application number
PCT/US1998/023265
Other languages
English (en)
Inventor
Jeffrey L. Sklar
Douglas T. Gjerde
Kimberly A. Lamb
Christopher P. Hanna
Original Assignee
Transgenomic, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/039,061 external-priority patent/US6355417B2/en
Priority claimed from US09/129,105 external-priority patent/US6287822B1/en
Application filed by Transgenomic, Inc. filed Critical Transgenomic, Inc.
Priority to EP98956451A priority Critical patent/EP1042503A4/fr
Priority to AU12975/99A priority patent/AU1297599A/en
Publication of WO1999023257A1 publication Critical patent/WO1999023257A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/366—Ion-pair, e.g. ion-pair reversed phase
    • 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 concerns a chromatographic method for detection of mutations in nucleic acids.
  • mutant and wild type DNA in the sample are very similar. In fact, their sequence may differ by only a single base pair. Therefore, the primers which would be used to amplify the mutant DNA would also amplify the wild type since both are present in the sample. As a result, the relative amounts of mutant and wild type DNA would not change.
  • cancer patients are monitored for the presence of residual cancer cells to determine whether the patients are in remission.
  • the effectiveness of these treatments can be monitored if small levels of residual cancer cells could be detected in a predominantly large wild type population.
  • the remission status is assessed by a pathologist who conducts histological examination of tissues samples.
  • these visual methods are largely qualitative, time-consuming, and costly. At best, the sensitivity of these methods permits detection of about 1 cancerous cell in 100
  • Gel based analytical methods can detect mutations in heteroduplex DNA strands under "partially denaturing" conditions.
  • the term “partially denaturing” means the separation of a mismatched base pair (caused by temperature, pH, solvent, or other known factors) in a DNA double strand while the remainder of the double strand remains intact.
  • these gel based techniques are operationally difficult to implement and require highly skilled personnel.
  • the analyses are lengthy and require a great deal of set-up time.
  • a denaturing capillary gel electrophoresis analysis of a 90 base pair fragment takes more than 30 minutes and a denaturing gel electrophoresis analysis may take 5 hours or more.
  • the long analysis time of the gel methodology is further exacerbated by the fact that the movement of DNA fragments in a gel is inversely proportional, in a geometric relationship, to their length. Therefore, the analysis time of longer DNA fragments can be often be untenable. Sample recovery of DNA fragments separated on a gel is difficult and time consuming, requiring specialized techniques.
  • the present invention is a method for detecting a putative mutant DNA in a sample of DNA, the method including the steps of (a) amplifying the sample of DNA using PCR, (b) hybridizing the amplified sample to form a mixture of homoduplexes and heteroduplexes, (c) separating the product of step (b) into fractions by Denaturing Matched Ion Polynucleotide Chromatography, and (d) blind collecting the fractions from step (c) at a retention time corresponding to the retention time of the heteroduplex.
  • the method preferably includes amplifying the fractions collected in step (d) using PCR to obtain an increased amount of heteroduplex relative to homoduplex.
  • the method can also include repeating steps (a) through (d); in a preferred method these steps are repeated until the relative amount of mutant to wild type DNA is increased by an enhancement factor of at least 10 to 1000.
  • the DNA sample can contain a large background of wild type.
  • the putative mutant DNA can be below the limit of detection.
  • the identity of the heteroduplex can be confirmed using standard methods.
  • the DNA sequence of the wild type DNA and the mutant DNA are known.
  • the mutant DNA differs from wild type DNA by at least one base pair.
  • the same PCR primers are used to amplify both the mutant DNA and the wild type DNA in the sample.
  • the retention time used in the blind collection of the heteroduplex in step (d) was previously determined from a reference standard.
  • a preferred reference standard is obtained by separating a standard mixture of homoduplex and heteroduplx, having the same base pair sequence as the sample, by Matched Ion Polynucleotide Chromatography.
  • the separation of the product by Denaturing Matched Ion Polynucleotide Chromatography is effected with an MIPC column containing a stationary phase separation media, and the column is treated before the separating step with a solution for removing any residual DNA from prior separations.
  • the column can be treated before the
  • the present invention is a method for screening a tissue sample for cancerous cells by detecting a putative mutant DNA in the DNA of the sample, the method including the steps of (a) amplifying the sample DNA using PCR, (b) hybridizing the amplified sample to form a mixture of homoduplexes and heteroduplexes, (c) separating the product of step (b) into fractions by Denaturing Matched Ion Polynucleotide Chromatography, and (d) blind collecting the fractions from step (c) at a retention time corresponding to the retention time of the heteroduplex.
  • the method preferably includes amplifying the fractions collected in step (d) using PCR to obtain an increased amount of heteroduplex relative to homoduplex.
  • the method can also include repeating steps (a) through (d); in a preferred method these steps are repeated until the relative amount of mutant to wild type DNA is increased by an enhancement factor of at least 10 to 1000.
  • the DNA sample can contain a large background of wild type.
  • the putative mutant DNA can be below the limit of detection.
  • the identity of the heteroduplex can be confirmed using standard methods.
  • the DNA sequence of the wild type DNA and the mutant DNA are known. In a typical analysis using the method of the invention, the mutant DNA differs from wild type DNA by at least one base pair.
  • the same PCR primers are used to amplify both the mutant DNA and the wild type DNA in the sample.
  • the retention time used in the blind collection of the heteroduplex in step (d) was previously determined from a reference standard.
  • a preferred reference standard is obtained by separating a standard mixture of homoduplex and heteroduplx, having the same base pair sequence as the sample, by Matched ion Polynucleotide Chromatography.
  • FIG. 1 is a schematic representation of hybridization of wild type DNA strand with homozygous mutant strand showing the production of two homoduplexes and two heteroduplexes.
  • FIG. 2 is a DMIPC chromatogram showing the separation of a standard mixture of FIG. 1.
  • FIG. 3 are DMIPC chromatograms demonstrating mutation detection using blind collection.
  • the present invention relates, therefore, to the unambiguous detection and identification of very small amounts of heteroduplex fragments containing mutant DNA in the presence of a relatively very large amount of known wild type using a recently developed chromatographic method called Denaturing Matched Ion Polynucleotide Chromatography (DMIPC), a method analogous to Matched Ion Polynucleotide Chromatography (MIPC). MIPC separates DNA fragments based on their base pair length (U.S.
  • Matched Ion Polynucleotide Chromatography is defined as a process for separating single and double stranded polynucleotides using non-polar separation media, wherein the process uses a counter-ion agent, and an organic solvent to release the polynucleotides from the separation media. MIPC separations are complete in less than 10 minutes, and frequently in less
  • MIPC systems WAVETM DNA Fragment Analysis System
  • MIPC uses unique non-polar separation media which comprises organic polymers, silica media having a non-polar surface comprising coated or covalently bound organic polymers or covalently bound alkyl and/or aryl groups, and continuous non-polar separation media, i.e., monolith or rod columns such as non-polar silica gel or organic polymer.
  • the separation media used in MIPC can be porous or non-porous.
  • MIPC systems and separation media are commercially available (Transgenomic, Inc. San Jose, CA).
  • the entire MIPC analysis can be automated by means of a desk top computer and a sample auto-injector. Analytical data for each sample can be analyzed in real time, or collected and stored in a computer memory device for analysis at a later time.
  • MIPC partially denaturing temperature
  • An important requirement for effective blind collections according to this invention is the absence from the separation media of any DNA fragments or other contaminants from prior separations.
  • One procedure for insuring this prerequisite is cleaning the column after each separation with a suitable cleaning
  • the present invention provides a method for detecting mutations in a sample containing a relatively large amount of wild type, wherein the concentration of the mutation is below the limits of detection a detector.
  • the invention provides a method for detecting mutations when the concentration of mutant DNA in a sample may be sufficient to detect, but the mutant DNA is not seen because it is obscured by the relatively large amount of wild type in the sample.
  • the invention takes advantage of the unique and surprising attributes of MIPC and DMIPC to accomplish the objective of detecting mutations in such samples, wherein the wild type and mutant are known.
  • the PCR primers are selected to yield fragments for which complete resolution of heteroduplexes from homoduplexes can be achieved by MIPC. Details for suitable primer selection are provided in copending U.S. Patent Application Serial No. 09/129,105 filed August 4, 1998, the entire contents of which are hereby incorporated by reference.
  • MIPC separates DNA fragments on the basis of their base pair length.
  • the method is highly reproducible. Therefore, columns do not have to be calibrated from sample to sample or from day to day. A DNA fragment of a particular base pair length will elute from an MIPC column at a specific retention time which is reliably reproducible. This characteristic, coupled with the automation, sample collection, and rapid sample analysis capabilities of MIPC make this method uniquely suited for detection of minute quantities of mutations in the presence of a large background of wild type.
  • blind collection is defined herein to mean the collection of mobile phase flowing through an MIPC column over a specific time interval subsequent to application of a DNA sample to the column. More specifically, “blind collection” refers to collecting mobile phase during the retention time interval corresponding to a previously determined retention time interval of a DNA fragment standard. Since the relationship between MIPC retention time and base pair length is highly reproducible, it is not necessary to detect a desired fragment with a detector in order to know when to collect the fragment. Column mobile phase is simply collected at the predetermined and expected retention time of a desired fragment.
  • the invention comprises a number of steps which eliminate any ambiguity regarding the presence or absence of a particular mutant fragment in a sample when the sample contains a large amount of wild type DNA relative to a putative mutation. These steps are described hereinbelow. Since the base sequence of the sample wild type DNA and the putative mutation are known, standards of these materials are combined and hybridized.
  • Hybridization is effected by heating the combined standards to about 90°C, then
  • the duplex strands in the sample denature, i.e., separate to form single strands. Upon cooling, the strands recombine. If a mutant strand was present in the sample having at least one base pair difference in sequence than wild type, the single strands will recombine to form a mixture of homoduplexes and heteroduplexes.
  • a standard mixture of homoduplexes and heteroduplexes is formed as depicted schematically in FIG. 1.
  • the standard mixture contains the same homoduplexes and heteroduplexes present in a sample which contains a putative mutation, albeit not in the same ratio.
  • This standard mixture cannot be separated by MIPC under normal conditions, since the heteroduplex and homoduplex have the same base pair length.
  • MIPC is performed at a temperature sufficiently elevated to selectively and partially denature a heteroduplex at the site of base pair mismatch (DMIPC)
  • the partially denatured heteroduplex will separate from a homoduplex having the same base pair length. Therefore, the hybridized standard mixture is applied to a MIPC column and a separation is performed under DMIPC conditions.
  • the chromatogram so produced shows a separation of the homoduplexes and heteroduplexes as shown in FIG. 2.
  • the retention times of the separated homoduplex and heteroduplex standards can then be used to predict the retention times of putative mutations having a concentration too low to be detected by a detector.
  • the retention times of the separated homoduplex and heteroduplex standards can then be used to predict the retention times of putative mutations in samples wherein the mutation signal is obscured by the wild type signal.
  • a sample containing a putative mutation is amplified using PCR to increase the total quantity of sample. Since the sequence is known, primers can be designed to maximize the fidelity of replication and minimize the formation of reaction artifacts and by-products. Approaches to primer design and PCR optimization for mutation detection by DMIPC are discussed in co-pending U.S. Patent Application 09/129,105 filed August 4, 1998.
  • wild type and mutant DNA strands in a sample have a nearly identical base sequence. A mutation may contain only one base pair difference compared to wild type. Therefore, primers cannot be designed to selectively anneal to, and preferentially amplify the mutant strand in the presence of wild type. Therefore, when such a sample is amplified using PCR, the ratio of mutant to wild type in the amplified product will be the same as in the original sample.
  • the amplified sample When the amplified sample is analyzed using MIPC a single major peak will be seen in the resulting chromatogram. This peak represents the combined wild type and mutant DNA, if the latter is present. No separation is achieved because the mutant and wild type DNA have the same base pair length. Therefore, the amplified sample is hybridized and analyzed under partially denaturing conditions by DMIPC. However, the heteroduplex corresponding to the putative mutation, if present, will not be seen by the detector either because its concentration is below the detection limits of the detector or because the ratio of wild type to putative mutation is very large so that the wild type homoduplex peak obscures the heteroduplex peak. In either case, the hetroduplex corresponding to the mutant DNA in the original sample need not be seen as a chromatographic peak to be determined.
  • the mobile phase is "blind collected” from the column at the expected retention time.
  • a tissue sample of at least about 100,000 cells is obtained for analysis. It is possible that, despite the initial DNA amplification, there will still be too little heteroduplex to detect. It is also possible that despite the separation of the homoduplex and heteroduplex, some homoduplex may have been collected along with the heteroduplex at the expected heteroduplex retention time, contaminating the heteroduplex and making it difficult to determine without ambiguity whether or not a mutation was present in the original sample. However, the ratio of homoduplex to heteroduplex will now be increased in favor of the heteroduplex compared to the ratio in the original sample.
  • the "blind collected" mobile phase described hereinabove preferably is concentrated, e.g., by evaporation of the mobile phase. If a mutation was present in the original sample, the residue will now be enriched in the heteroduplex. This heteroduplex enriched residue is amplified again by PCR and the products are hybridized. The hybridized products of the second PCR amplification will now contain an increased amount of heteroduplex relative to homoduplex. This process is described in Example 1 and depicted in FIG. 3.
  • the evaporation can be effected with standard and conventional DNA solution evaporation equipment, for example, the SPEEDVAC evaporator (Model UCS 100 Universal Speed Vac system, Savant Instruments, Inc, Hayward, CA)
  • the steps comprising the method of the invention were designed to enrich the sample in heteroduplex in order to enable the detection of mutations which would normally go undetected.
  • the steps of the method of the invention can be reiterated a plurality of times to increase the purity and quantity of heteroduplex to any desired level.
  • the increased amount of heteroduplex compared to homoduplex obtained in this manner can be described by an "enhancement factor".
  • the “enhancement factor” is defined herein as the increase in the ratio of heteroduplex to homoduplex compared to the ratio of heteroduplex to homoduplex in the original hybridized sample, wherein the increase results from the implementation of the method of the invention.
  • the “enhancement factor” depends on the number of iterations performed and can range from 10 to more than 1 ,000.
  • the PCR product is hybridized and analyzed by DMIPC. If the original sample contained a mutation, the concentration of heteroduplex or its concentration relative to wild type, will now be sufficient to detect. The DMIPC chromatogram will, therefore, show a peak having the retention time of the standard heteroduplex. In this event it can be concluded unambiguously that a mutation was present in the original sample.
  • an aliquot of standard heteroduplex can be mixed with an aliquot of the heteroduplex enriched sample.
  • a DMIPC chromatogram of this mixture will show an increase in the area of the heteroduplex peak, compared to the area of the heteroduplex enriched sample peak alone.
  • the purification and enrichment method described above will provide sufficient heteroduplex for determination of its base pair sequence.
  • Denaturing gradient gel electrophoresis techniques which can separate homoduplexes from heteroduplexes cannot be used as an alternative to DMIPC. Although samples can be recovered form gels with difficulty, blind collection is not possible because the mobility of a DNA fragment in a gel is not constant.
  • the detection of cancer cells in early diagnosis screens or in evaluations of a cancer treatment regimen is usually about 1 cancer cell in 100 total cells, or
  • the present invention increases the sensitivity of cancer cell detection to about 1 cancer cell in 100,000 total cells.
  • the presence of cancer cells can be detected down to a level of about 0.001%. The tremendous extension of the lower limits for cancer cell detection made possible by this invention can save countless lives.
  • homoduplex peaks at a retention time of about 6.5 minutes. No heteroduplex can be seen.
  • Another aliquot of the same sample was chromatographed on the same column and mobile phase was collected between 4.5 and 6.3 minutes. The mobile phase was evaporated to dryness, and the residue was amplified using standard PCR techniques.
  • the lower trace of the DMIPC chromatogram shown in FIG. 3 now shows a previously undetected heteroduplex peak at a retention time of about 6.2 minutes.
  • Solvent A 0.1 M triethylammonium acetate (TEAA)
  • Solvent B 25% acetonitrile in 0.1 M TEAA

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Abstract

Un procédé permettant de détecter un ADN mutant présumé dans un échantillon d'ADN comprend les étapes suivantes: l'amplification de l'échantillon d'ADN par PCR; l'hybridation de l'échantillon amplifié pour former un mélange d'homoduplexes et d'hétéroduplexes; la séparation du mélange en fractions par Chromatographie d'Ions Appariés Dénaturants (MIPC); la récupération aveugle des fractions éluées après un temps de rétention correspondant au temps de rétention de l'hétéroduplex. L' ADN présent dans les fractions collectées de manière aveugle peut être amplifié par PCR pour obtenir une plus grande quantité d'hétéroduplex par rapport à l'homoduplex. Ce procédé est utile pour déterminer l'état de rémission d'un patient dont l'échantillon d'ADN provenant de tissus contient un fond important de type sauvage ou dans lequel l'ADN mutant présumé se situe en-deçà du seuil de détection.
PCT/US1998/023265 1997-10-31 1998-10-30 Procede de detection d'adn mutant par mipc et pcr WO1999023257A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98956451A EP1042503A4 (fr) 1997-10-31 1998-10-30 Procede de detection d'adn mutant par mipc et pcr
AU12975/99A AU1297599A (en) 1997-10-31 1998-10-30 Method of detecting mutant dna by mipc and pcr

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US6443797P 1997-10-31 1997-10-31
US60/064,437 1997-10-31
US09/039,061 1998-03-13
US09/039,061 US6355417B2 (en) 1997-03-14 1998-03-13 Band array display of polynucleotide separations
US5858098A 1998-04-10 1998-04-10
US5833798A 1998-04-10 1998-04-10
US09/129,105 US6287822B1 (en) 1997-08-05 1998-08-04 Mutation detection method
US10331398P 1998-10-06 1998-10-06
US09/129,105 1998-10-06
US09/058,337 1998-10-06
US09/058,580 1998-10-06
US60/103,313 1998-10-06

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AU (1) AU1297599A (fr)
WO (1) WO1999023257A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094366A1 (fr) * 2000-06-02 2001-12-13 Blue Heron Biotechnology, Inc. Methode visant a ameliorer la fidelite sequentielle des oligonucleotides synthetiques bicatenaires
GB2371048A (en) * 2001-01-10 2002-07-17 Univ York Assay for determining allelic variations in prion protein genes
WO2001095233A3 (fr) * 2000-06-02 2003-03-20 Transgenomic Inc Analyse de donnees issues de la separation chromatographique en phase liquide d'adn
EP1278584B1 (fr) * 2000-04-21 2006-11-15 TRANSGENOMIC, Inc. Procede de lavage de colonne par chromatographie de polynucleotides a ions apparies
EP1798291A1 (fr) * 2005-12-13 2007-06-20 Institut Curie Méthode pour la détection de mutations dans les acides nucléiques, et leurs utilisation dans la diagnose des maladies genétiques et des cancers

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US5585236A (en) * 1992-11-18 1996-12-17 Sarasep, Inc. Nucleic acid separation on alkylated nonporous polymer beads
US5795976A (en) * 1995-08-08 1998-08-18 The Board Of Trustees Of The Leland Stanford Junior University Detection of nucleic acid heteroduplex molecules by denaturing high-performance liquid chromatography and methods for comparative sequencing

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US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) * 1985-03-28 1990-11-27 Cetus Corp
US5585236A (en) * 1992-11-18 1996-12-17 Sarasep, Inc. Nucleic acid separation on alkylated nonporous polymer beads
US5795976A (en) * 1995-08-08 1998-08-18 The Board Of Trustees Of The Leland Stanford Junior University Detection of nucleic acid heteroduplex molecules by denaturing high-performance liquid chromatography and methods for comparative sequencing

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Title
HUBER C. G., ET AL.: "HIGH-RESOLUTION LIQUID CHROMATOGRAPHY OF OLIGONUCLEOTIDES ON NONPOROUS ALKYLATED STYRENE-DIVINYLBENZENE COPOLYMERS.", ANALYTICAL BIOCHEMISTRY., ACADEMIC PRESS INC., NEW YORK., vol. 212., 1 January 1993 (1993-01-01), NEW YORK., pages 351 - 358., XP002916339, ISSN: 0003-2697, DOI: 10.1006/abio.1993.1340 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1278584B1 (fr) * 2000-04-21 2006-11-15 TRANSGENOMIC, Inc. Procede de lavage de colonne par chromatographie de polynucleotides a ions apparies
WO2001094366A1 (fr) * 2000-06-02 2001-12-13 Blue Heron Biotechnology, Inc. Methode visant a ameliorer la fidelite sequentielle des oligonucleotides synthetiques bicatenaires
WO2001095233A3 (fr) * 2000-06-02 2003-03-20 Transgenomic Inc Analyse de donnees issues de la separation chromatographique en phase liquide d'adn
US6664112B2 (en) 2000-06-02 2003-12-16 Blue Heron Biotechnology, Inc. Methods for improving the sequence fidelity of synthetic double-stranded oligonucleotides
GB2371048A (en) * 2001-01-10 2002-07-17 Univ York Assay for determining allelic variations in prion protein genes
EP1798291A1 (fr) * 2005-12-13 2007-06-20 Institut Curie Méthode pour la détection de mutations dans les acides nucléiques, et leurs utilisation dans la diagnose des maladies genétiques et des cancers

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EP1042503A1 (fr) 2000-10-11
EP1042503A4 (fr) 2002-01-16

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