Sequencing of nucleic acids
First Claim
1. A method of sequencing a double stranded nucleic acid, comprising:
- (a) ligating adaptors to said nucleic acid, wherein said adaptors include double stranded oligonucleotide sequence which incorporates a predetermined nuclease recognition sequence for a nuclease whose recognition site is displaced from its cleavage site, said displacement being such as to create, as a result of said litigation, cleavage sites in the resulting ligation products which, upon cleavage thereat, result in removal of a base or bases from one strand of said nucleic acid, and wherein at least some of said adaptors have double stranded oligonucleotide sequence which incorporates at least two different nuclease recognition sequences;
(b) cleaving litigation products from (a) with said nuclease to produce double stranded products having a single stranded oligonucleotide extension;
(c) subjecting said products from (b) to ligation with a population of adaptors which include double stranded oligonucleotide sequence having extending single strands, wherein said population of adaptors includes molecules having in their extending single strands permutations, optionally all possible such permutations, of a base or bases constituting a predetermined number of bases, and wherein each permutation is provided with a respective unique and detectable label, each adaptor in said population having a nuclease recognition sequence for a nuclease whose recognition site is displaced from its cleavage site, said displacement being such as to create, as a result of the ligation of this step (c) cleavage sites in the resulting ligation products which, upon cleavage thereat, result in removal of a base or bases from one strand of each said products from (b);
(d) separating the ligation products from (c);
(e) cleaving the separated ligation products from (d) with the nuclease of (c) to produce a population of fragments carrying the recognition site of nuclease of (c);
(f) either analysing the labels carried by ligation products separated in (d), or analysing the labels carried by fragments from (e); and
(g) repeating steps (c) to (f) as often as necessary to determine the desired sequence, but with the final repeat optionally omitting step (e).
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Abstract
The present invention provides a method of sequencing a nucleic acid, comprising either sequentially removing bases from the sequence of the nucleic acid a predetermined number at a time, with the product remaining from each step of predetermined base removal being ligated to a labeled adapter specific for said bases and including oligonucleotide sequence, or hybridizing a primer to the nucleic acid to be sequenced and sequentially extending said primer a predetermined number of bases at a time, said added base(s) being complementary to base(s) in the nucleic acid being sequenced, and each of said base addition steps being achieved by the use of a labeled adaptor specific for said bases and including oligonucleotide sequence containing said predetermined base(s); in either case, the label of said labeled adaptor being specific for its respective predetermined base(s).
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Citations
13 Claims
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1. A method of sequencing a double stranded nucleic acid, comprising:
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(a) ligating adaptors to said nucleic acid, wherein said adaptors include double stranded oligonucleotide sequence which incorporates a predetermined nuclease recognition sequence for a nuclease whose recognition site is displaced from its cleavage site, said displacement being such as to create, as a result of said litigation, cleavage sites in the resulting ligation products which, upon cleavage thereat, result in removal of a base or bases from one strand of said nucleic acid, and wherein at least some of said adaptors have double stranded oligonucleotide sequence which incorporates at least two different nuclease recognition sequences;
(b) cleaving litigation products from (a) with said nuclease to produce double stranded products having a single stranded oligonucleotide extension;
(c) subjecting said products from (b) to ligation with a population of adaptors which include double stranded oligonucleotide sequence having extending single strands, wherein said population of adaptors includes molecules having in their extending single strands permutations, optionally all possible such permutations, of a base or bases constituting a predetermined number of bases, and wherein each permutation is provided with a respective unique and detectable label, each adaptor in said population having a nuclease recognition sequence for a nuclease whose recognition site is displaced from its cleavage site, said displacement being such as to create, as a result of the ligation of this step (c) cleavage sites in the resulting ligation products which, upon cleavage thereat, result in removal of a base or bases from one strand of each said products from (b);
(d) separating the ligation products from (c);
(e) cleaving the separated ligation products from (d) with the nuclease of (c) to produce a population of fragments carrying the recognition site of nuclease of (c);
(f) either analysing the labels carried by ligation products separated in (d), or analysing the labels carried by fragments from (e); and
(g) repeating steps (c) to (f) as often as necessary to determine the desired sequence, but with the final repeat optionally omitting step (e). - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method of sequencing, a double-stranded nucleic acid having a nucleotide sequence comprising sequentially removing nucleotide bases from said nucleotide sequence of the nucleic acid a predetermined number at a time, ligating the product remaining from each step of predetermined nucleotide base removal to a labelled adapter specific for said bases said adaptor including oligonucleotide sequence, containing a predetermined bases(s), the label of said labelled adaptor being specific for its respective predetermined bases(s);
- wherein at least some of said adaptors have double stranded oligonucleotide sequence which incorporates at least two different nuclease recognition sequences.
- View Dependent Claims (9, 10, 11, 12, 13)
Specification