EP0356021A2
Expired / Lapsed
EP
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A method for the amplification of nucleotide sequences
Assignee: Syngenta Ltd
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Filing: 1989-07-27
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Granted: —
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Expires: 2009-07-27
Abstract
Abstract A method for the amplification of a nucleic acid fragment containing unknown sequence and kits therefor are provided which enable long nucleotide sequences to be rapidly and efficiently sequenced. The method comprises the formation of target nucleic acid fragment/vectorette units by cleavage of target nucleic acid followed by ligation. One of the nucleic acid fragments will contain an initiating priming region of known sequence for hybridisation with-an initiating primer and target nucleic acid fragment/vectorette units will contain a vectorette priming region of known sequence for hybridisation with a vectorette primer. Amplification is effected by primer extension of an initiating primer hybridised to the initiating priming region of the target nucleic acid fragment/vectorette unit.
Inventors
- 👤 Alexander Fred Markham
- 👤 John Craig Smith
- 👤 Rashida Anwar
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 19:34
CPC Classifications
C; C12; C12N; C12N15/00; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C07; C07H; C07H21/00; C08; C08G; C08G77/00; C08G77/42; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/6844; C12Q1/6853; C12Q1/6855; C12Q1/6883; C12Q1/6888; C12Q1/689; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
171 words
A method for the amplification of a nucleic acid fragment, comprising unknown sequence, by primer extension which method comprises cleaving a target nucleic acid to obtain target nucleic acid fragments, one of said fragments containing an initiating priming region of known nucleotide sequence for hybridisation with an initiating primer, preparing target nucleic acid fragment/vectorette units from the target nucleic acid fragments by ligation each unit having a vectorette priming region of known sequence for hybridisation with a vectorette primer, and treating the target nucleic acid fragment/vectorette units, together or sequentially, with appropriate nucleoside triphosphates and an agent for polymerisation of nucleoside triphosphates under hybridising conditions, such that an extension product of an initiating primer is synthesised complementary to a single stranded target nucleic acid/vectorette unit having an initiating priming region to which is hybridised an initiating primer selected so as to be substantially complementary to the initiating priming region, whereas no such extension product is synthesised complementary to single stranded target nucleic acid fragment/vectorette units having no such initiating priming region.
Claim 2
Dependent Claim
37 words
A method as claimed in claim 1 wherein the extension product is subjected to amplification in the presence of a vectorette primer which primer is selected so as to be substantially complementary to a vectorette priming region.
Claim 3
Dependent Claim
29 words
A method as claimed in claim 2 wherein the synthesis of vectorette primer amplification products is dependent upon the initial synthesis of an extension product of the initiating primer.
Claim 4
Dependent Claim
94 words
A method as claimed in claim 3 wherein the vectorette portion of the target nucleic acid fragment/vectorette unit comprises a double stranded portion having first and second strands, the second strand of the vectorette portion being ligated to that strand of the target nucleic acid fragment which contains the initiating priming region, and the nucleotide sequence of the first strand, second strand and vectorette primer being selected such that the vectorette primer is capable of hybridising to the complement of the second strand but not to the first strand under the same hybridisation conditions.
Claim 5
Dependent Claim
113 words
A method as claimed in claim 3 wherein the vectorette portion of the target nucleic acid fragment/vectorette unit comprises a double stranded portion having first and second strands, the first strand having a terminal polymerisation blocking moeity and the second strand, which in use is ligated to that strand of the target nucleic acid fragment containing the initiating priming region, carrying a single stranded portion, the terminal polymerisation blocking moiety being effective to prevent extension of the first strand to form a complement to the said single stranded portion of the second strand in the presence of appropriate nucleoside triphosphates and an agent for the polymerisation of the nucleoside triphosphates under hybridising conditions.
Claim 6
Independent Claim
97 words
A method as claimed in any one of the preceding claims wherein a plurality of different vectorette libraries is prepared for use with the same single initiating primer, each vectorette library comprising target nucleic acid fragment/vectorette units obtained by ligation of nucleic acid fragments prepared by cleaving target nucleic acid at different cleavage sites; treating each vectorette library either separately or together with appropriate nucleoside triphosphates and an agent for polymerisation of nucleoside triphosphates under hybridising conditions whereby to obtain a plurality of initiating primer extension products, based on the use of the same single initiating primer.
Claim 7
Dependent Claim
127 words
A method as claimed in claim 1 or claim 2 wherein a nucleic acid having termini capable of ligation to each other is circularised, the nucleic acid containing a portion there of being capable of serving as an initiating priming region for hybridisation with an initiating primer; cleaving the circularised nucleic acid outside the known nucleotide sequence to form a linear molecule containing the known nucleotide sequence and having a known cleavage site pattern at at least one terminus for ligation to form a target nucleic acid fragment/vectorette unit; forming said target nucleic acid fragment/vectorette unit by ligation and treating the target nucleic acid fragment/vectorette unit, together or sequentially, with initiating primer, appropriate nucleoside triphosphates and an agent for polymerisation of the nucleoside triphosphates under hybridising conditions.
Claim 8
Dependent Claim
36 words
A method as claimed in claim 7 wherein the target nucleic acid fragment/vectorette unit is treated, together or sequentially with initiating primer, vectorette primer, appropriate nucleoside triphosphates and an agent for polymerisation of the nucleoside triphosphates.
Claim 9
Independent Claim
60 words
A method as claimed in any one of the preceding claims wherein the target nucleic acid fragment/vectorette units are prepared from target nucleic acid fragments by ligation such that the vectorette cannot be cleaved from the target nucleic acid fragment/vectorette units obtained therefrom by the same agent used to cleave the target nucleic acid to yield target nucleic acid fragments.
Claim 10
Dependent Claim
58 words
A method as claimed in claim 9 wherein the target nucleic acid is cleaved with a restriction endonuclease to yield a target nucleic acid fragment for ligation to a vectorette, the sequence of the vectorette being selected such that the restriction endonuclease recognition sequence of the said restriction endonuclease is absent in the target nucleic acid fragment/vectorette unit.
Claim 11
Independent Claim
64 words
A method as claimed in any one of the preceding claims wherein any or all of the initiating primer extension products obtained is (are) sequenced at least at the end(s) distal to a given initiating primer so as to determine the sequence of a further initiating primer whereby to obtain further initiating primer extension products based on primer extension of the further initiating primer.
Claim 12
Independent Claim
32 words
A method as claimed in any one of the preceding claims wherein an initiating primer extension product or portion thereof is sequenced whereby to characterise the said extension product or portion thereof
Claim 13
Dependent Claim
52 words
A method as claimed in claim 11 or claim 12 for the identification of a genotype responsible for a given phenotype wherein the sequence of nucleic acid containing the genotype is compared with the sequence of nucleic acid containing no such genotype whereby to identify the genotype responsible for the given phenotype.
Claim 14
Dependent Claim
30 words
A method as claimed in claim 13 wherein the comparison is effected by the use of a single sample of nucleic acid from an obligate heterozygote for the given phenotype.
Claim 15
Dependent Claim
96 words
A method as claimed in claim 11 or claim 12 for the identification of a genotype responsible for or contributory to a predisposition to a given phenotype if such be present wherein the sequences of nucleic acid from a plurality of individuals affected by the phenotype to be investigated are compared with the sequences of nucleic acid from a plurality of individuals in which no evidence of the phenotype to be investigated is present whereby to identify a genotype responsible for or contributory to a predisposition to the phenotype to be investigated if such be present.
Claim 16
Dependent Claim
53 words
A method as claimed in claim 15 wherein the sequence differences between 1) a pool of nucleic acids from a plurality of individuals affected by the phenotype to be investigated; and 2) a pool of nucleic acids from a plurality of individuals presenting no evidence of the phenotype to be investigated; are compared.
Claim 17
Dependent Claim
111 words
A kit for the amplification of a nucleic acid fragment of unknown sequence by primer extension, which kit comprises:- 1) means for cleaving a target nucleic acid at a specific site to obtain a target nucleic acid fragment; 2) a vectorette adapted for ligation to a target nucleic acid fragment obtained by use of the means for cleaving a target nucleic acid defined in (1) whereby to form in use a target nucleic acid fragment/vectorette unit said vectorette having a vectorette priming region of known sequence for hybridisation with a vectorette primer; 3) each of four different nucleoside triphosphates; and 4) an agent for polymerisation of the nucleoside triphosphates in (3).
Claim 18
Dependent Claim
41 words
A kit as claimed in claim 12 which additionally comprises a vectorette primer and if desired at least one nested primer, said primer(s) having a nucleotide sequence substantially complementary to a vectorette priming region of a target nucleic acid fragment/vectorette unit.
Claim 19
Dependent Claim
98 words
A kit as claimed in claim 17 or claim 18 wherein the vectorette adapted for ligation to a target nucleic acid fragment comprises a double stranded portion having first and second stands, the second strand of the vectorette being adapted for ligation to that strand of the nucleic acid fragment which contains the initiating priming region, and the nucleotide sequence of the first strand and second strand being selected such that in use a vectorette primer is capable of hybridising to the complement of the second strand but not to the first strand under the same hybridisation conditions.
Claim 20
Dependent Claim
75 words
A vectorette library kit for the amplification of a nucleic acid fragment of unknown sequence by primer extension, which kit comprises:- 1) at least one vectorette library each library comprising a set of target nucleic acid fragment/vectorette units obtained from nucleotide sequences of an individual member of a species of animal, plant or microorganism; and 2) an initiating primer or primers for hybridisation to an initiating priming region of the target nucleic acid fragment/vectorette units.
Claim 21
Dependent Claim
79 words
A kit as claimed in claim 20 for the analysis of a genotype responsible for or contributory to a predisposition to a given phenotype wherein the set of target nucleic acid fragment/vectorette units comprises:- 1) a pool of target nucleic acid fragment/vectorette units from a plurality of individuals affected by the phenotype to be investigated; and 2) a pool of target nucleic acid fragment/vectorette units from a plurality of individuals presenting no evidence of the phenotype to be investigated.
References Cited (Backward Citations)
No citation records indexed.
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