WO2003070977A2 - Procede permettant de detecter des polymorphismes a nucleotide unique - Google Patents

Procede permettant de detecter des polymorphismes a nucleotide unique Download PDF

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Publication number
WO2003070977A2
WO2003070977A2 PCT/EP2003/001420 EP0301420W WO03070977A2 WO 2003070977 A2 WO2003070977 A2 WO 2003070977A2 EP 0301420 W EP0301420 W EP 0301420W WO 03070977 A2 WO03070977 A2 WO 03070977A2
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WIPO (PCT)
Prior art keywords
sequence
nucleotide sequences
atc
sample
nucleic acid
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PCT/EP2003/001420
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English (en)
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WO2003070977A3 (fr
Inventor
Andreas Kappel
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Nanogen Recognomics Gmbh
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Priority to AU2003210259A priority Critical patent/AU2003210259A1/en
Publication of WO2003070977A2 publication Critical patent/WO2003070977A2/fr
Publication of WO2003070977A3 publication Critical patent/WO2003070977A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a method which can be used for detecting mutations, in particular Single Nucleotide Polymorphisms, by enzymatic processing in parallel in different nucleic acids on a solid surface.
  • the major goal of pharmacogenomic research will be the continuous discovery of genetic variations within the human population, and the subsequent correlation of these variations with particular phenotypes. This may allow to develop better diagnostic tools, safer medications, and will help to study the biological functions of genes.
  • Large-scale sequencing projects are in progress, that aim to draw a map of the most frequently occuring Single Nucleotide Polymorphisms (SNPs), the type of mutation that is expected to contribute most to the genetic variation within the human population.
  • SNPs Single Nucleotide Polymorphisms
  • mismatch repair proteins of the mutS family use the ability of mismatch repair proteins of the mutS family to bind to mutant DNA.
  • mismatches are generated by the hybridisation of a DNA that serves as a reference sequence, with the complementary sequence from a different individual; if the two sequences differ, a mismatch is generated, which is discriminated by stronger mutS binding (Jiricny, J., Su, J.J., Wood, S.G. & Modrich, P. Mismatch-containing oligonucleotide duplexes bound by the E.co///77ttfS-encoded protein. Nucl. Acids Res. 16, 7843- 7853 (1988); Taylor, G.R. & Deeble, J. Enzymatic methods for mutation scanning. Genetic Analysis: Biomolecular Engineering 14, 181 -186 (1999)).
  • US 5,556,750 and US 5,922, 539 describe methods for eliminating DNA duplex molecules containing base pair mismatches by removing molecules with single stranded regions or by degradation of cleaved heterohybrids.
  • WO 96/41002 describes methods for mismatch dependent DNA sequencing and positional cloning.
  • all these methods are not adaptable to a parallel working microarray format, e.g. because of the necessary separation, degradation or sequencing of cleaved or gapped DNA strands, and therefore they are not compatible to high-throughput diagnostic platforms for the parallel analysis of multiple patients with high specificity.
  • the invention is based on the object of making available a new method for detecting mutations, in particular Single Nucleotide Polymorphisms (SNPs) in nucleotide sequences, which permits in particular a high sample throughput in a short time and with a high degree of reliability.
  • SNPs Single Nucleotide Polymorphisms
  • dNTPs desoxynucleotidetriphosphates
  • the 5' -ends of the sample nucleic acid sequence and of the reference nucleic acid sequence are protected.
  • the 5'- ends could be blocked by substituting the 5' -phosphate group by a thiol group during oligonucleotide synthesis.
  • reference nucleotide sequence denotes a nucleotide sequence, preferably a DNA sequence, which is used as a comparison sequence
  • sample nucleotide sequence is a labeled nucleotide sequence, preferably a DNA sequence, which is to be examined for mutations.
  • the d(G methyl-A T C) containing sample nucleotide sequences and the reference sequences are obtainable by amplifying a DNA sample with known sequence by PCR for the preparation of the reference nucleic acid sequence and amplifying a DNA sample with unknown sequence for the preparation of the sample nucleotide sequence wherein a primer containing a d(G Me ATC) sequence is used.
  • Typical primers are polymers of 15 to 35 nucleotides.
  • the hybridization of the d(G Me ATC) containing sample nucleotide sequences with single-stranded reference nucleotide sequences can be carried out as described in Frederic N., Ausubel, Short Protocols in Molecular Biology, Section 2.10, Wiley + Sons.
  • microelectronic arrays allow a site-specific and individualized hybridisation of nucleic acid strands.
  • they are ideal tools for the generation of heteroduplex nucleic acids by first addressing a biotinylated reference nucleic acid with known sequence to an individual test site, and subsequently addressing a labelled, complementary sample nucleic acid from e.g. a patient sample to the same surface. If the sample DNA contains a mutation when compared to the reference DNA, the heteroduplex will contain a mismatch. This mismatch is site- specific detectable by the present method using the mutS, mutL and mutH recognition and incision system.
  • the fixing of the single-stranded or double-stranded nucleotide sequences, and the hybridization can be electronically controlled, in particular electronically accelerated.
  • a particularly preferred embodiment of the claimed method is characterized by a site-resolved, electronically accelerated hybridization, with the hybridization conditions, such as the current strength applied, the voltage applied or the duration of the electronic addressing, being set individually at the respective site.
  • the base mispairing can be incised by adding mutS which recognizes mispairings, mutL and mutH which incises the d(G Me ATC) sequence complementary region of the reference nucleic acid.
  • the electronic addressing is effected by applying an electric field, preferably between 1.5 V and 2.5 V in association with an addressing duration of between 1 and 3 minutes. Due to the electric charge on the nucleotide sequences to be addressed, their migration is greatly accelerated by an electric field being applied.
  • the addressing can be effected in a site-resolved manner; in this case, addressing takes place consecutively to different zones on the chip surface. At the same time, different addressing and hybridization conditions can be set at the individual sites.
  • nucleotide sequence heteroduplexes consisting of a predetermined nucleotide sequence, i.e. the reference nucleotide sequence, and of the complementary nucleotide sequence from a physiological sample, i.e. the sample nucleotide sequence, are initially produced on a chip surface using electronic addressing.
  • the mispairings which are formed in this connection indicate a mutation in the sample nucleotide sequence and can be incised using mutS which binds to the mispairing site, mutL and mutH which incise the d(G Me ATC) sequence complementary region of the reference nucleic acid.
  • Base mispairing-binding mutS, mutL and mutH are derived e.g. from E.coli, T. thermophilus or T.aquaticus.
  • the reference nucleotide sequence for example, can be employed as a biotinylated oligonucleotide which is either synthesized or prepared by amplification using sequence-specific oligonucleotides, one of which is biotinylated at the 5' end.
  • the reference nucleotide sequence is converted into the single-stranded state by melting, preferably in a buffer solution having a low salt content, and applied to a predetermined position on a chip by means of electronic addressing. Examples of suitable chips are those marketed by Nanogen (San Diego/USA).
  • the reference nucleotide sequence can be applied, for example, using a Nanogen molecular biology workstation, preferably using the parameters specified by the manufacturer. Unless otherwise indicated, Nanogen's chips and/or their molecular biology workstation is/are used in accordance with the manual which is supplied with them; the method of use is also described in Radtkey et al., Nucl. Acids Res. 28, 2000, e17.
  • the sample nucleotide sequence which is complementary to the sequence which has already been applied to the chip, can now be loaded onto the chip which has been prepared in this way.
  • dye-labeled and d(G Me ATC)-labeled oligonucleotides are synthesized or generated by amplifying the sample nucleotide acid sequence using sequence-specific oligonucleotides one of which is dye-labeled at the 5' end.
  • the dye-labeled sample nucleotide sequence constitutes the complementary strand to the biotinylated strand of the reference nucleotide sequence.
  • the sample nucleotide sequence has also to be converted beforehand into the single-stranded state by being melted, for example in a buffer solution having a low salt content, and then applied to the biotinylated reference nucleotide sequence by means of electronic addressing.
  • the heteroduplex can also be prepared on an electronically addressable surface, for example using a Nanogen molecular biology workstation and employing the parameters specified by the manufacturer. Successful hybridization can be monitored optically, and at the same time determined quantitatively, by detecting the dye which is coupled to the heteroduplex.
  • sample nucleotide sequence can also be biotinylated and electronically addressed, as just described. It is also possible to hybridize in solution, with subsequent electronic addressing and with one of the two nucleotide sequences of the heteroduplex being biotinylated. Apart from derivatizing with biotin, it is also possible to use other molecular groups, which bind to an electronically addressable surface, for fixing nucleotide sequences. Thus, it is likewise possible, for example, to effect the fixing using introduced thiol groups, hydrazine groups or aldehyde groups.
  • the support material comprises in a preferred embodiment a permeation layer.
  • the permeation layer is selected from the group consisting of silicon, silicon dioxide, silicon nitride, controlled porosity glass, metal, metal silicilide, inorganic sol-gels, and hydrogels.
  • Preferred hydrogels are selected from the group consisting of agarose, polyacrylamide, polymethacrylamide, and organic polymer hydrogels.
  • mutS which recognizes these mispairings with a high degree of specificity, is used for binding such mispairings and mutL and mutH are added to incise the heteroduplex which is recognized by mutS.
  • the mispairing- recognizing mutS proteins derived from E.coli and from T. thermophilus are particularly suitable for this purpose.
  • the mispairing-recognizing mutS, mutL and the incising mutH are preferably added in excess.
  • the reaction is carried out at low salt conditions, concentration of from 10 to 300 mM salt concentration, in particular from 10 to 150 mM is preferred.
  • the 3 ' -ends of the incised reference nucleic acids are removed. Removing of said 3 ' -ends is preferably carried out by increasing the temperature, by washing with low ionic strength or by an enzymatic digestion with a 5'>3' exonuclease, such as lambda exonuclease or T7 exonuclease.
  • the polymerization is carried out by adding a 3 >5 and 5 " >3 X exonuclease deficient DNA polymerase such as Klenow fragment 3'>5' exo ⁇ and by applying desoxynucleotidetriphosphates (dNTPs), wherein at least one of said dNTPs contains a detectable label.
  • dNTPs desoxynucleotidetriphosphates
  • the DNA polymerization takes specifically place on the prior mismatched heteroduplexes.
  • the addition of a 3'>5' exonuclease deficient DNA polymerase is sufficient, if 5' protected sample and reference nucleic acids are hybridized. (Polymerization and digestion can be carried out as described in Frederic N., Ausubel, Short Protocols in Molecular Biology, Section 3.5 and 3.11 , Wiley + Sons.)
  • the labeling moiety of the labeled dNTPs is selected from the group consisting of fluorescent moieties, visible dye moieties, radioactive moieties, chemiluminescent moieties.
  • the resulting labeled heteroduplexes are detectable by fluorescence, chemiluminescence, visible light or radioactive radiation scanning.
  • An advantage of the present method is the reliability concerning the detection of mispairings, based on the mutH endonuclease which incises the reference nucleic acid region complementary to the methylated d(G e ATC) sequences in amplified DNA with high specificity and activity.
  • the use of the mismatch repair system will allow to detect mismatches with a greater sensitivity than with labeled mutS alone.
  • An additional advantage is that the mutS protein binds to dsDNA containing a mismatch at any position of the DNA sequence and mutS protein binds all kinds of mismatches (A:A, A:G, A:C, G:G, G:T, C:C, C:T, T:T).
  • Another important advantage of the present method is the possibility of simultaneously detecting mutations in parallel, in an integrated manner in one procedure, in particular on an active electronic array.
  • the electronic addressing can be effected, for example, on a chip, on which the nucleotide sequences A, B, C..., N are already fixed at sites a, b, c to n, using a mixture containing nucleotide sequences from the group A' , B' , C N' .
  • the nucleotide sequences A/A' to N/N' in each case constitute a reference and sample nucleotide sequence pair.
  • the stringency of the hybridization conditions can be increased, for example, by reversing the polarity of the electrical field. This can be effected in a site-resolved manner and consequently be adjusted individually in the case of each site.
  • one major benefit of the active electric microarray technology is that several individuals can be analyzed on the same chip, which is not possible on passive microarrays.
  • Another embodiment of the present invention are the d(G Me ATC) containing nucleic acids for the detection of mutations, e. g. for the detection of SNPs, in particular on an active electronic microarray.
  • a further embodiment are the d(G Me ATC) containing primers for the preparation of the d(G Me ATC) containing sample nucleic acid sequences by PCR.
  • Figure 1 describes a preferred method for detecting mutations in nucleotide sequences. Therefore the reference nucleic acid (step (a)) and a sample nucleic acid (step (b)) are prepared by PCR. The PCR of step (b) is carried out with d(G Me ATC) containing forward primers. The single-stranded, d(G Me ATC) containing sample nucleotide sequences and the corresponding single-stranded reference nucleotide sequences are hybridized (step (c)).
  • the resulting heteroduplex is incubated with mutS, mutL and mutH (mutSLH) wherein the exonuclease mutH is bound through mutL and the mismatch recognizing mutS to the mismatch containing heteroduplex (step (d)) and the hemimethylated heteroduplex is incised by the mutH exonuclease at the reference nucleic acid region which is complementary to the methylated d(G Me ATC)-sequence of the sample nucleic acid (step (e)).
  • mutS mutS, mutL and mutH
  • step (f) The 3' -end of the incised reference nucleic acids are removed (step (f)) and the hybridized sample nucleotide sequences are incubated with desoxynucleotidetriphosphates (dNTPs), wherein at least one of said dNTPs contains a detectable label (black circles (step (g)), and with a 3'>5' and 5'>3' exonuclease deficient DNA polymerase (step (g)).
  • dNTPs desoxynucleotidetriphosphates
  • Figure 2 shows a similar process as described in Figure 1 wherein the 5' -ends of the reference nucleic acid sequence and the sample nucleic acid sequence are protected. After the incision of the mismatch containing hetereoduplex the 3' -end of the reference nucleic acid sequence is removed by applying a 5'>3'- exonuclease (step (f)) followed by the incubation step g) as described in Fig. 1 , wherein the addition of only a 3'>5' exonuclease deficient DNA polymerase is sufficient.

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Abstract

L'invention concerne un procédé pouvant être utilisé pour détecter des mutations en parallèle dans des polymorphismes particuliers à nucléotide unique dans différentes séquences nucléotidiques. Ladite méthode comprend les étapes procédurales suivantes: hybridation de séquences nucléotidiques échantillons à brin unique contenant d(GMeATC) avec des séquences nucléotidiques de référence à brin unique; fixation de séquences nucléotidiques de référence à brin unique ou de séquences nucléotidiques échantillons à brin unique avant ou pendant l'hybridation, ou d'hétéroduplex constitués de séquences nucléotidiques de référence et de séquences nucléotidiques échantillons après ou pendant l'hybridation sur une surface; incubation avec mutS, mutL et mutH afin d'inciser la région complémentaire de la séquence d(GMeATC) dans la séquence d'acides nucléiques de référence, lorsqu'il existe un appariement défectueux dans les séquences d'acides nucléiques de référence et échantillons hybridées; et suppression des extrémités 3' des acides nucléiques de référence incisées et incubation avec une ADN ploymérase et des desoxynucléotidetriphosphates marqués.
PCT/EP2003/001420 2002-02-21 2003-02-13 Procede permettant de detecter des polymorphismes a nucleotide unique WO2003070977A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005030041A2 (fr) 2003-09-25 2005-04-07 Third Wave Technologies, Inc. Detection de papillomavirus
FR2920159A1 (fr) * 2007-08-20 2009-02-27 Serial Genetics Sas Soc Par Ac Methode et reactifs pour la quantification, la comparaison et l'identification d'alleles sur support solide
WO2013074632A1 (fr) * 2011-11-14 2013-05-23 Rose Floyd D Purification et identification de nucléotides mésappariés

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WO1995012688A1 (fr) * 1993-01-11 1995-05-11 United States Biochemical Corporation Procedes d'analyse et de manipulation d'adn a l'aide de systemes de reparation d'erreurs d'appariement
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WO1996041002A2 (fr) * 1995-06-07 1996-12-19 Genzyme Corporation Methode d'identification de modification genetique d'adn consistant en un sequencage d'adn et un clonage de position
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EP1041160A1 (fr) * 1997-07-31 2000-10-04 The Institute Of Physical & Chemical Research Procede de detection de mutation dans une sequence de bases
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US5376526A (en) * 1992-05-06 1994-12-27 The Board Of Trustees Of The Leland Stanford Junior University Genomic mismatch scanning
WO1995012688A1 (fr) * 1993-01-11 1995-05-11 United States Biochemical Corporation Procedes d'analyse et de manipulation d'adn a l'aide de systemes de reparation d'erreurs d'appariement
WO1996023903A1 (fr) * 1995-01-30 1996-08-08 Ulf Landegren Procede de detection de variations dans des sequences d'acide nucleique
WO1996041002A2 (fr) * 1995-06-07 1996-12-19 Genzyme Corporation Methode d'identification de modification genetique d'adn consistant en un sequencage d'adn et un clonage de position
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WO1999045153A2 (fr) * 1998-03-05 1999-09-10 The University Of Iowa Research Foundation Methode iterative et regenerative de sequencage de l'adn
DE10038237A1 (de) * 2000-08-04 2002-02-14 Aventis Res & Tech Gmbh & Co Verfahren zum Nachweis von Mutationen in Nucleotidsequenzen

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SMITH J ET AL: "MUTATION DETECTION WITH MUTH, MUTL, AND MUTS MISMATCH REPAIR PROTEINS" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 93, 1 April 1996 (1996-04-01), pages 4374-4379, XP002030021 ISSN: 0027-8424 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005030041A2 (fr) 2003-09-25 2005-04-07 Third Wave Technologies, Inc. Detection de papillomavirus
EP1664348A2 (fr) * 2003-09-25 2006-06-07 Third Wave Technologies, Inc. Detection de papillomavirus
EP1664348A4 (fr) * 2003-09-25 2007-01-10 Third Wave Tech Inc Detection de papillomavirus
US7527948B2 (en) 2003-09-25 2009-05-05 Third Wave Technologies, Inc. Detection of HPV
US8389245B2 (en) 2003-09-25 2013-03-05 Third Wave Technologies, Inc. Detection of HPV
US8765418B2 (en) 2003-09-25 2014-07-01 Third Wave Technologies, Inc. Detection of HPV
FR2920159A1 (fr) * 2007-08-20 2009-02-27 Serial Genetics Sas Soc Par Ac Methode et reactifs pour la quantification, la comparaison et l'identification d'alleles sur support solide
WO2013074632A1 (fr) * 2011-11-14 2013-05-23 Rose Floyd D Purification et identification de nucléotides mésappariés

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