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1. WO1996006187 - NUCLEOTIDE SEQUENCING METHOD

Publication Number WO/1996/006187
Publication Date 29.02.1996
International Application No. PCT/GB1995/001987
International Filing Date 21.08.1995
Chapter 2 Demand Filed 14.03.1996
IPC
C12Q 1/68 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING 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
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
68involving nucleic acids
CPC
C12Q 1/6858
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
68involving nucleic acids
6844Nucleic acid amplification reactions
6858Allele-specific amplification
C12Q 1/6869
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
68involving nucleic acids
6869Methods for sequencing
Applicants
  • THE SOLICITOR FOR THE AFFAIRS OF HER MAJESTY'S TREASURY IN HER BRITANNIC MAJESTY'S GOVERNMENT OF THE UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND [GB/GB]; Queen Anne's Chambers, 28 Broadway London SW1H 9JS, GB (AllExceptUS)
  • TULLY, Gillian [GB/GB]; GB (UsOnly)
  • SULLIVAN, Kevin [GB/GB]; GB (UsOnly)
Inventors
  • TULLY, Gillian; GB
  • SULLIVAN, Kevin; GB
Agents
  • SKELTON, S., R.; Ministry of Defence (PE) Directorate of Intellectual Property Rights Poplar Building 2a, MOD Abbey Wood #19 PO Box 702 Bristol BS 12 7DU, GB
Priority Data
9417211.125.08.1994GB
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) NUCLEOTIDE SEQUENCING METHOD
(FR) PROCEDE DE SEQUENÇAGE DE NUCLEOTIDES
Abstract
(EN)
A method is provided for determining the identity of at least two discrete single nucleotide bases each adjacent to a predetermined nucleotide base sequence in a target sample comprising one or more types of polynucleotide chain, the method comprising incorporating a capture group into the target sample and immobilising the target sample by means of the capture group; mixing the target sample with (i) nucleotide primers which are complementary to the characterised base sequences such that they anneal thereto at positions adjacent to the bases to be identified and (ii) at least two types of dideoxynucleoside triphosphate (ddNTP), each type labelled with a distinguishable fluorescent group, and (iii) a nucleotide chain extending enzyme such that ddNTPs complementary to the bases to be identified are incorporated into the nucleotide primers; eluting the extending nucleotide primers, separating the types of extended nucleotide primer on basis of size or charge and identifying the ddNTPs incorporated into each type of nucleotide primer by reference to the fluorescent characteristics associated with the distinguishable groups. Primers and a test kit are provided for use in performance of the method.
(FR)
On décrit un procédé qui permet de déterminer l'identité d'au moins deux bases nucléotidiques uniques distinctes, dont chacune est adjacente à une séquence de bases nucléotidiques prédéterminée, dans un échantillon cible comprenant un ou plusieurs types de chaînes polynucléotidiques. Ce procédé consiste à incorporer un groupe de capture dans l'échantillon cible et à immobiliser ce dernier avec ce groupe, à mélanger cet échantillon (i) avec des amorces nucléotidiques complémentaires des séquences de base à caractères définis, de façon qu'elle s'hybrident à ces séquences en des positions adjacentes aux bases à identifier et (ii) avec au moins deux types de didésoxynucléosides triphosphates (ddNTP) marqués chacun avec un groupe fluorescent distinct et (iii) avec une enzyme d'extension de chaîne nucléotidique de façon que les ddNTP complémentaires des bases à identifier soient incorporés aux amorces nucléotidiques. Il consiste ensuite à élever les amorces nucléotidiques étendues puis à les séparer par tailles ou par charges et à identifier les ddNTP incorporés dans chaque type d'amorce nucléotidique par référence aux caractéristiques de fluorescence associées aux groupes pouvant être distingués. On décrit aussi des amorces et une trousse d'essai permettant de mettre ce procédé en ÷uvre.
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