WO2003072814A2 - Amelioration de la methode de pcr specifique d'allele - Google Patents

Amelioration de la methode de pcr specifique d'allele Download PDF

Info

Publication number
WO2003072814A2
WO2003072814A2 PCT/EP2003/001725 EP0301725W WO03072814A2 WO 2003072814 A2 WO2003072814 A2 WO 2003072814A2 EP 0301725 W EP0301725 W EP 0301725W WO 03072814 A2 WO03072814 A2 WO 03072814A2
Authority
WO
WIPO (PCT)
Prior art keywords
primer
discriminating
terminal
proxi
sequence variant
Prior art date
Application number
PCT/EP2003/001725
Other languages
English (en)
Other versions
WO2003072814A3 (fr
Inventor
Alfred Pingoud
Meinhard Hahn
Björn Tews
Jochen Wilhelm
Peter Friedhoff
Andreas Marx
Michael Strerath
Daniel Summerer
Original Assignee
Roche Diagnostics Gmbh
F.Hoffmann-La Roche Ag
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
Application filed by Roche Diagnostics Gmbh, F.Hoffmann-La Roche Ag filed Critical Roche Diagnostics Gmbh
Priority to AU2003229549A priority Critical patent/AU2003229549A1/en
Publication of WO2003072814A2 publication Critical patent/WO2003072814A2/fr
Publication of WO2003072814A3 publication Critical patent/WO2003072814A3/fr

Links

Classifications

    • 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

Definitions

  • the present invention lies in the field of detecting nucleic acid sequence variants with the aid of PCR.
  • the invention concerns an improved method for allele- specific amplification using modified primers.
  • nucleic acid variants such as mutations or single nucleotide polymorphisms.
  • a variant of a target nucleic acid can be detected by hybridizing the nucleic acid sample to be analysed with a sequence variant-specific hybridization probe under suitable hybridization conditions.
  • allele-specific amplification is an alternative that is already known in the prior art for sequence variant-specific hybridization.
  • variant-specific amplification primers are already used during the amplification which usually have a discriminating terminal nucleotide residue at the 3' terminal end of the primer which is only complementary to a special variant of the target nucleic acid to be detected.
  • US 5,595,890 for example describes such methods for allele-specific amplification and their use to detect clinically relevant point mutations for example in the k-ras oncogene.
  • US 5,521,301 also describes methods for allele-specific amplification for genotyping the ABO blood group system.
  • US 5,639,611 discloses the use of allele-specific amplification in connection with the detection of the point mutation responsible for sickle cell anaemia.
  • Such methods for detecting sequence variants, polymorphisms and, above all, point mutations require an allele-specific amplification particularly when the sequence variant to be detected is present in a lower amount compared to a variant of the same section of nucleic acid (or of the same gene) that is present in excess.
  • This situation for example occurs when the aim is to detect disseminating tumour cells in body fluids such as blood, serum or plasma with the aid of allele-specific amplification (US 5,496,699).
  • the DNA is firstly isolated from body fluids such as blood, serum or plasma which is composed of a relatively small amount of DNA from disseminating tumour cells and an excess of DNA from non- proliferating cells.
  • mutations in the k-Ras gene that are significant for tumoral DNA have to be detected on the basis of a few copies of tumoral DNA in the presence of an excess of wild type DNA.
  • the object of the present was to provide a sequence variant-specific detection method which allows a sequence variant to be detected with increased specificity.
  • Another object of the present invention was to provide a detection method in which the discriminating nucleotide residue is not necessarily at the 3' end of the discriminating primer.
  • This object is achieved according to the invention by a method for determining the presence or absence of at least one sequence variant in one or more target nucleic acids in an individual sample characterized by a sequence variant-specific amplification reaction in which the sequence variant to be detected in the target nucleic acid is complementary to the 3' terminal, 3' proxi-terminal or 3'-proxi-proxi- terminal nucleotide residue of the discriminating primer, essentially characterized in that one or more nucleotide residues of at least one discriminating primer are substituted at the 4' position of the (deoxy) ribose.
  • This substituent is preferably a C1-C6 alkyl, alkenyl or alkinyl residue, where the alkenyl residue may be monounsaturated or polyunsaturated.
  • Methyl, ethyl or vinyl groups are particularly preferred as substituents.
  • the substituted nucleotide residue is identical to the discriminating nucleotide residue and is located at the terminal or proxi- terminal position of the discriminating primer.
  • substituted nucleotide residue is present at the terminal position and the discriminating nucleotide residue is present at the proxi-terminal position of the discriminating primer.
  • the invention concerns a method for determining the presence or absence of at least one sequence variant in one or more target nucleic acids in an individual sample comprising the following steps:
  • an agent for the template-dependent polymerization of the deoxynucleoside triphosphates for example a DNA polymerase
  • At least one discriminating primer containing at least one discriminating nucleotide residue, one primer being added for each sequence variant of a target nucleic acid to be detected, said primer having a sequence which is complementary to the sequence variant to be detected, and the sequence variant to be detected in the target nucleic acid being complementary to at least one 3' terminal, 3' proxi- terminal or 3' proxi-proxi-terminal nucleotide residue of the discriminating primer,
  • nucleotide residues are substituted by at least one discriminating primer at the 4' position of the (deoxy) ribose
  • At least one additional primer which is complementary to a primer extension product which is formed by extension of a discriminating primer
  • a target nucleic acid is a section of a nucleic acid from a biological sample whose sequence is to be analysed in more detail with the aid of the method according to the invention.
  • the biological sample is usually composed of genomic DNA.
  • the method according to the invention can be used equally well to analyse RNA sequence variants. It is unimportant whether the sample has been isolated from cellular material or biological liquids such as blood, serum, plasma, urine or saliva.
  • a sequence variant in the sense of the invention means a target nucleic acid having a particular nucleic acid sequence which differs only minimally from sequences of other possible target nucleic acids and which is to be distinguished from these.
  • the differences in the sequence preferably concern between one and three consecutive nucleotide residues.
  • the present invention is particularly suitable for identifying sequence variants concerning only a single nucleotide residue. These variants can be a base exchange or alternatively nucleotide additions or deletions. Various alleles can be distinguished from one another in this manner. Point mutations or polymorphisms can also be detected.
  • a sequence variant is also to be understood in particular as point mutations or polymorphisms which are analysed for the purposes of obtaining prognostic or diagnostic information.
  • a template-dependent polymerization of deoxynucleotide triphosphates begins at the 3' end of a so-called primer which is hybridized to a single-stranded template nucleic acid resulting in a polymerization of the deoxynucleotide triphosphates in such a manner that a sequence complementary to the target nucleic acid is formed.
  • Such polymerization reactions in the 5' - 3' orientation are preferably carried out enzymatically using so-called DNA polymerases such as Klenow polymerase.
  • Thermostable DNA polymerases such as Taq polymerase (Roche Applied Science catalogue No. 1146165) are particularly preferred.
  • primer extension Reactions in which a complementary primer bound to a target nucleic acid is enzymatically extended are also referred to as primer extension.
  • DNA-dependent DNA polymerases or RNA-dependent DNA polymerases are advantageously used for this.
  • thermostable polymerases which have no 3'-5' proofreading activity which is the case for Taq polymerase or vent exo " polymerase (New England Biolabs).
  • the template-dependent polymerization is a reverse transcriptase reaction which can be carried out with the reverse transcriptases known to a person skilled in the art.
  • a discriminating primer in the sense of the invention is a primer whose sequence is exactly complementary to a particular sequence variant where this sequence has certain differences to another sequence variant which may be present in the sample to be analysed.
  • a discriminating nucleotide residue means a nucleotide residue whose complement is formed by different nucleotide residues in the various existing sequence variants.
  • the 3'-terminal nucleotide residue is the nucleotide residue which is located at the terminal end of an oligonucleotide primer and has a free 3'-OH group.
  • the proxi- terminal nucleotide residue is the nucleotide residue of an oligonucleotide primer whose end is linked via a phosphate group to the 5' end of the terminal nucleotide residue.
  • Proxi-proxi-terminal nucleotide residue refers to the nucleotide residue whose 3' end is linked via a phosphate group to the 5' end of the proxi-terminal nucleotide residue.
  • steps a) — e) are essentially an amplification reaction which leads to an amplification product depending on the presence or absence of a particular sequence reaction. Such methods can therefore be carried out according to known protocols for PCR reactions from the prior art.
  • kits containing agents for carrying out the method according to the invention.
  • a kit contains a first discriminating primer containing at least one discriminating nucleotide residue wherein this primer is substituted at the 4' position of the (deoxy)ribose and at least one other primer which is complementary to a primer extension product formed by extension of a discriminating primer.
  • kit can optionally contain additional components such as deoxynucleotide triphosphate and agents for the template-dependent polymerization of the deoxynucleotide triphosphates such as a DNA polymerase.
  • a DNA polymerase is preferably thermostable.
  • the individual components of the kit can, in various embodiments, optionally be contained in a common storage vessel or separately in two or more storage vessels.
  • the primer containing 4'-(deoxy)ribose-modified nucleotide residues which enable a sequence variant-specific amplification according to the invention
  • the effect of a potential steric hindrance may depend on the polymerase that is used in a particular case.
  • the 4' residue on the (deoxy)ribose of the nucleotide residues has the function of influencing the structure of the primer/target double- strand hybrid or its interaction with the polymerase.
  • nucleotide residue means a nucleotide monomer building block which is a constituent of the oligonucleotides.
  • ribose usually means 2'-deoxyribose but can also mean ribose or (deoxy)ribose analogues in specific embodiments of the invention.
  • Preferred residues for the 4'-(deoxy)ribose substitution are: alkyl, alkenyl, alkinyl, aryl, heteroaryl, cycloalkyl,
  • alkyl in which alkyl is linear or branched, can optionally contain a heteroatom and is Cl- C6, alkenyl, alkinyl is C2-C6 and linear or branched, aryl is C6-C10, cycloalkyl is C3- C6.
  • substituents are 4' methyl, ethyl or vinyl residues.
  • nucleobases the only limitation is that a base pairing must be possible by means of hydrogen bridges i.e. the oligonucleotides that are used can, in addition to the naturally occurring bases, also contain analogues thereof.
  • Analogues in connection with the invention are nucleosides having other heterocycles as bases (Claire, S., In Advanced Chemistry Tests, Nucleoside Mimetics (2001) chapters 4, 6, edited by Gordon and Breach Science Publishers, Amsterdam) such as aza analogues of the natural bases in which a CH unit (methine carbon atom) of the purine or pyrimidine ring is replaced by a nitrogen, (such as 8-aza-A) and base analogues in which a ring nitrogen atom is replaced by a CH group (such as 7-deaza G), or combinations of aza and deaza substitutions (such as 8 aza, 7 deaza G).
  • C-nucleosides are the deaza forms in which the nitrogen N-9 in the . purine series or the nitrogen Nl in the pyrimidine series is replaced by an sp2 carbon atom as is for example the case for formycin and pseudouridine (Claire, S., in Advanced Chemistry texts, Nucleoside Mimetics (2001) chapters 4, 6, edited by Gordon and Breach Science Publishers, Amsterdam). Bases with a modified hydrogen donor/hydrogen acceptor pattern such as 2 amino A and inosine can also be used as base analogues. The listed nucleobases and analogues can also be substituted in which case the substituent should not hinder formation of hydrogen bonds with the complementary base. Preferred positions are C5 in the case of pyrimidines, C8 in the case of purines and C7 in the case of deazapurines.
  • the oligonucleotides used for the invention can also contain other modified monomers in addition to a 4'-modified monomer building block. These other modified monomers can be modified on the sugar such as e.g. a 4' modification as in the case of LNA. The modification can also be a substitution on a base such as e.g. 5- propinyl U or a replacement of a natural base by an analogue such as 7 deaza G or nitroindole. Oligonucleotides can also contain a label or a reactive group as a further modification.
  • the oligonucleotide can also contain a non-nucleosidic linker at the 5' terminus or internally in which case the linker may be provided with a label or a reactive group.
  • the phosphate backbone of the oligonucleotides may also be modified e.g. contain one or more phosphothioate bridges between the monomers instead of phosphates. Chimers of various backbone modifications can also be used within the scope of the present invention.
  • the design of the primer according to the invention is limited with regard to the position of the discriminating nucleotide residue as well as with regard to the position of the labelled nucleotide residue within the discriminating primer.
  • the substituted nucleotide residue is located at the 3'- terminal, 3'-proxi-terminal or 3'-proxi-proxi- terminal position of the discriminating primer.
  • the discriminating nucleotide residue is located at the 3'-terminal, 3' proxi-terminal or 3' proxi-proxi-terminal position of the primer. This does not in anyway exclude the possibility that the discriminating nucleotide residue may e identical to the substituted nucleotide residue.
  • connection figure 1 shows a schematic representation of six preferred embodiments of the invention in which X is the respective discriminating nucleotide residue and Sub represents the respective substituted nucleotide residue.
  • alternatives a) and c) have the advantage that the substituted nucleotide residue is located at the 3' end of the oligonucleotide primer and thus does not have to be introduced into the oligonucleotide in the form of an expensive commercial phosphoramidite, but instead can be coupled before the start of the oligonucleotide synthesis to a suitable CPG solid phase and hence such a synthesis is relatively cheap.
  • alternatives b), d) and f) have the advantage that when the substituted nucleotide residue according to the invention is located at the proxi-terminal position of the primer this enables the best results to be obtained with regard to the specificity of the claimed method.
  • the method according to alternative e) also surprisingly leads to highly specific results.
  • the amplification products can be detected by electrophoretic methods. This has the disadvantage that under certain conditions an amplification product is formed which can be detected by gel electrophoresis even in the case of a low extension rate of inventively modified primers hybridized to a mismatch.
  • a further embodiment of the present invention concerns the procedure for the method according to the invention with the aid of real time PCR.
  • the time course of the amplification reaction is monitored with the aid of suitable fluorescent-labelled hybridization probes which enables kinetic real time measurements that allow an analysis of the amplification products that are generated with the lowest possible number of cycles.
  • the hybridization probes that are used for the method according to the invention are usually single-stranded nucleic acids such as single-stranded DNA or RNA or derivatives thereof or alternatively PNAs, which hybridize with the target nucleic acid at the annealing temperature of the amplification reaction.
  • These oligonucleotides usually have a length of 20 to 100 nucleotides.
  • the label can be introduced at any desired (deoxy)ribose or phosphate group of the oligonucleotide depending on the exact detection format. Labels at the 5' and 3' end of the nucleic acid molecule are preferred. It must be possible to detect this type of label in the real time mode of the amplification reaction. This is for example not only possible using fluorescent labels but alternatively also with the aid of labels that can be detected with the aid of NMR.
  • test procedures can be employed.
  • the following three detection formats have proven to be particularly suitable in connection with the present invention:
  • Two single-stranded hybridization probes are used simultaneously with this test format which are complementary to adjacent sites of the same strand of the amplified target nucleic acid. Both probes are labelled with different fluorescent components. When excited with light of a suitable wavelength, a first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer such that when both hybridization probes are bound to adjacent positions of the target molecule to be detected, a fluorescence emission of the second component can be measured.
  • a single-stranded hybridization probe is labelled with two components.
  • the first component is excited with light of a suitable wavelength, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer.
  • the hybridization probe binds to the target DNA and is degraded by the 5'-3' exonuclease activity of Taq polymerase during the subsequent elongation phase.
  • the excited fluorescent component and the quencher are spatially separated from one another such that a fluorescence emission of the first component can be measured.
  • hybridization probes are also labelled with a first component and with a quencher and the labels are preferably located at the two ends of the probe.
  • the two components are in spatial proximity due to the secondary structure of the probe.
  • the two components are separated from one another and hence the fluorescence emission of the first component can then be measured after excitation with light of a suitable wavelength (US 5,118,801).
  • the respective amplification product can also be detected according to the invention by a DNA binding dye which interacts with double- stranded nucleic acid and emits an appropriate fluorescence signal after excitation with light of a suitable wavelength.
  • a DNA binding dye which interacts with double- stranded nucleic acid and emits an appropriate fluorescence signal after excitation with light of a suitable wavelength.
  • the dyes SybrGreen and SybrGold have proven to be particularly suitable for this application. It is also possible to alternatively use intercalating dyes.
  • Example 2 Coupling of N 4 -benzoyl-l-r5'-(4,4-dimethoxytrityl -4'-vinyl-2'-deoxy1- cytosine-3'-succinyl ester (19) to lcaa-cpg
  • Long chain alkyl amino controlled pore glass (lcaa-cpg) is placed in a second flask together with 109 ⁇ l triethylamine in 1.6 ml dimethylformamide.
  • the yellow suspension is freed from precipitated urea with the aid of a membrane filter and added to the suspended glass carrier. This results in an intensive bright yellow colouration of the suspension.
  • the suspension of the glass carrier is carefully shaken overnight for the coupling reaction.
  • the solid phase is purified over a frit (washed with 20 ml each of dimethylformamide, methanol and diethyl-ether).
  • the purified glass carrier is dried in a high vacuum and subsequently suspended in 2 ml pyridine.
  • the oligonucleotides according to the invention were synthesized on a 1 ⁇ mol scale.
  • Commercially available standard phosphoramidites (DMTr ibu G, DNTr bzA; DMTr bz C and DMTR T) and standard chemicals for synthesis from Glen Research were used for this.
  • the 4'-modified cytosine monomers were introduced with the aid of a CPG.
  • the 4'-modified thymidine monomers were introduced according to Summerer, D et al., supra and with the aid of a phosphoramidite.
  • the cleavage from the carrier and the cleavage of the protective groups was carried out using 33 % NH 3 for 8 h at 55°C.
  • the 5' DMTr oligonucleotide was purified by reversed phase (RP 18) chromatography: buffer A 0.1 M trie hylammonium acetate in water pH 7.0 /MeCN 95:5, buffer B: MeCN. Gradient 3 min 20 % B; 12 min 12- 40 % B, flow rate 1 ml/min, detection 260 nm.
  • the concentrated oligonucleotide solution was then treated for 5 min at room temperature with 2.5 % dichloroacetic acid / methylene chloride in order to remove the 5' DMTr protective group. Afterwards the oligonucleotides were desalted on an RP 18 column and subsequently lyophilized with the aid of a SpeedVac.
  • T* T H , T me
  • reaction buffer (0.10 M Tris-HCl pH 9.0, 10 mM MgCl 2 ), heated for 2 min to 95°C and subsequently cooled within 1 hour to room temperature.
  • 5 ⁇ l dNTP mkture (2 mM TTP, dATP, dGTP, dCTP each) 10 ⁇ l was removed from each reaction mixture as a blank value and added to 30 ⁇ l of a mixture of formamide / water (4:1) and 20 mM EDTA (stop solution). 2.5 units of enzyme were added at 72°C to the mixtures. Aliquots of 10 ⁇ l were removed after 1, 2, and 5 min and each was mixed with 30 ⁇ l of the above stop solution. The reaction products were denatured by heating to 95°C, separated by means of 14 % PAGE and analysed by Phosphor Imaging.
  • T* T me , T et
  • reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % Triton), heated for 2 min to 95°C and subsequently cooled within 1 hour to room temperature.
  • Example 7 PCR amplification using modified and non-modified primers
  • 0.5 pmol 90mer template strand 90A or 90G and 50 pmol primer 20T and 50 pmol antisense primer were dissolved in 50 ⁇ l reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 2 M MgSO , 0.1 % Triton) and 0.2 mM dNTP mkture were mked with 4 enzyme units of Vent(exo ' ) DNA polymerase.
  • Example 8 Dynamic range of the real time PCR using modified and non-modified primers
  • 10 7 copies of double-stranded DNA templates (146A, 146C, 146G or 146T) were amplified in a total of 10 ⁇ l of a reaction mkture having the following composition: 0.5 ⁇ M forward primer (F25T), 0.5 ⁇ M reverse primer (R20), 0.2 mM dNTP mixture, 0.5 g/1 BSA, 5.9 mM MgCl 2 , SYBR-Green in a 10 "3 dilution, 10 mM Tris-HCl, 50 mM KC1 and 0.5 U Taq DNA polymerase, pH 8.3 at 20°C.
  • a reaction mkture without template DNA served as a negative control.
  • the reaction components were mked at 4°C
  • measuring point end of the extension phase.
  • the resulting amplification curves for amplification with unmodified and modified primers are shown in fig. 7a) andb).
  • the figure shows that under the said assay conditions a discrimination of the mismatch (T/C, T/G, T/T) compared to the match (T/A) is only possible using the modified primer.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode et une trousse permettant de déterminer la présence ou l'absence d'au moins une variante de séquence dans un ou plusieurs acides nucléiques cibles dans un prélèvement individuel contenant : des désoxyribonucléosides triphosphates ; un agent servant à effectuer la polymérisation dépendant de la matrice des désoxyribonucléosides triphosphates, par exemple une ADN polymérase ; au moins une amorce discriminante contenant au moins un résidu nucléotidique discriminant, une amorce étant ajoutée pour chaque variante de séquence d'un acide nucléique cible à détecter, l'amorce ayant une séquence complémentaire à la variante de séquence à détecter, la variante de séquence à détecter dans l'acide nucléique cible étant complémentaire à au moins un résidu nucléotidique 3'-terminal, 3' proxi-terminal ou 3' proxi-proxi-terminal de l'amorce discriminante, laquelle invention se caractérise en ce qu'un ou plusieurs résidus nucléotidiques d'au moins une amorce discriminante sont substitués à la position 4' du (désoxy)ribose ; ainsi qu'au moins une amorce supplémentaire qui est complémentaire à un produit d'extension d'amorce formé par extension d'une amorce discriminante.
PCT/EP2003/001725 2002-02-26 2003-02-20 Amelioration de la methode de pcr specifique d'allele WO2003072814A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003229549A AU2003229549A1 (en) 2002-02-26 2003-02-20 Improved method for allele-specific pcr

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP02004221.4 2002-02-26
EP02004221 2002-02-26

Publications (2)

Publication Number Publication Date
WO2003072814A2 true WO2003072814A2 (fr) 2003-09-04
WO2003072814A3 WO2003072814A3 (fr) 2003-12-18

Family

ID=27763335

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/001725 WO2003072814A2 (fr) 2002-02-26 2003-02-20 Amelioration de la methode de pcr specifique d'allele

Country Status (2)

Country Link
AU (1) AU2003229549A1 (fr)
WO (1) WO2003072814A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2069497A2 (fr) * 2006-11-02 2009-06-17 University of Utah Research Foundation Oligonucleotides a utiliser en pcr specifique d'un allele
WO2011069677A1 (fr) * 2009-12-11 2011-06-16 Roche Diagnostics Gmbh Amplification spécifique à un allèle d'acides nucléiques
WO2011104695A3 (fr) * 2010-02-26 2012-03-01 GAMMAGENETICS Sàrl Détection de la mutation de kras dans l'exon 2 par rcp quantitative en temps réel spécifique aux allèles

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5639611A (en) * 1988-12-12 1997-06-17 City Of Hope Allele specific polymerase chain reaction
US5750343A (en) * 1993-12-09 1998-05-12 Syntex Inc. Methods of detecting nucleic acids with nucleotide probes containing 4'-substituted nucleotides and kits therefor
WO1999040219A1 (fr) * 1998-02-05 1999-08-12 Bavarian Nordic Research Institute A/S Quantification par inhibition de l'amplification

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5639611A (en) * 1988-12-12 1997-06-17 City Of Hope Allele specific polymerase chain reaction
US5750343A (en) * 1993-12-09 1998-05-12 Syntex Inc. Methods of detecting nucleic acids with nucleotide probes containing 4'-substituted nucleotides and kits therefor
WO1999040219A1 (fr) * 1998-02-05 1999-08-12 Bavarian Nordic Research Institute A/S Quantification par inhibition de l'amplification

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SUMMERER D AND MARX A: "Differential minor groove interactions between DNA polymerase and sugar backbone of primer and template strands" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 124, no. 6, 13 February 2002 (2002-02-13), pages 910-911, XP002213302 cited in the application *
SUMMERER D UND MARX A: "DNA polymerase selectivity: sugar interactions monitored with high-fidelity nucleotides" ANGEWANDTE CHEMIE, vol. 40, no. 19, May 2001 (2001-05), pages 3693-3695, XP002213303 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2069497A2 (fr) * 2006-11-02 2009-06-17 University of Utah Research Foundation Oligonucleotides a utiliser en pcr specifique d'un allele
EP2069497A4 (fr) * 2006-11-02 2010-08-04 Univ Utah Res Found Oligonucleotides a utiliser en pcr specifique d'un allele
US8580945B2 (en) 2006-11-02 2013-11-12 University Of Utah Oligonucleotides for use in allele-specific PCR
WO2011069677A1 (fr) * 2009-12-11 2011-06-16 Roche Diagnostics Gmbh Amplification spécifique à un allèle d'acides nucléiques
CN102656278A (zh) * 2009-12-11 2012-09-05 霍夫曼-拉罗奇有限公司 核酸的等位基因特异性扩增
US9238832B2 (en) 2009-12-11 2016-01-19 Roche Molecular Systems, Inc. Allele-specific amplification of nucleic acids
CN106987624A (zh) * 2009-12-11 2017-07-28 霍夫曼-拉罗奇有限公司 核酸的等位基因特异性扩增
WO2011104695A3 (fr) * 2010-02-26 2012-03-01 GAMMAGENETICS Sàrl Détection de la mutation de kras dans l'exon 2 par rcp quantitative en temps réel spécifique aux allèles

Also Published As

Publication number Publication date
WO2003072814A3 (fr) 2003-12-18
AU2003229549A1 (en) 2003-09-09

Similar Documents

Publication Publication Date Title
EP0628051B1 (fr) Utilisation de n-nucleosides fluorescents et de leurs analogues structurels fluorescents
JP4558932B2 (ja) ハイブリダイゼーション及び不一致識別のための、ピラゾロ［３，４−ｄ］ピリミジン含有オリゴヌクレオチド
EP1198594B1 (fr) Extension par polymerase a l'extremite 3' de chimere de pna-adn
CA2329135C (fr) Hybridation et discrimination du defaut de complementarite au moyen d'oligonucleotides conjugues a des molecules de liaison de petits sillons (mgb)
JP4360904B2 (ja) 単一ヌクレオチドの増幅およびポリメラーゼによる検出
EP2689031B1 (fr) Analogues de 3-alcynyl pyrazolopyrimidines fonctionnalisées comme bases universelles et procédés d'utilisation
EP1842923B1 (fr) Dérivés de méthanesulfonylaminoindole et sondes oligonucléotidiques marquées les contenant
US20080038745A1 (en) Nucleotide analogs with six-membered rings
WO2004071155A2 (fr) Sequençage en phase solide
JP2009017882A (ja) 標的ハイブリダイゼーション検出のための、プローブおよびクランプのバイナリー組成物ならびに方法
WO2008085652A1 (fr) Amplification par ligature
CN104011224A (zh) 用于降低非特异性扩增的方法和试剂
US6063571A (en) Process for amplifying nucleic acids using DNA/PNA primers
US7118871B2 (en) Reagents for monitoring nucleic acid amplification and methods of using same
EP1384789B1 (fr) Sondes d'hybridation fluorescentes ayant un bruit de fond réduit
JP4643262B2 (ja) 検体検出法
CN101671672B (zh) 用于改进的核酸扩增的聚阴离子
WO2003072814A2 (fr) Amelioration de la methode de pcr specifique d'allele
US7759469B2 (en) Labeling reagent
JP4643263B2 (ja) 対立遺伝子特異的プライマー伸長
JP3942079B2 (ja) 核酸増幅時の外因性コントロール、内部コントロールのための方法
EP1700922B1 (fr) Dérivés de 5-nitroindole 3-substitués et sondes oligonucléotidiques les contenant
US20030175769A1 (en) Reagents for improved PCR
EP1466919A1 (fr) Analogues nucleotidiques hexacycliques
WO2001023611A2 (fr) Procedes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP