Patent ID: 6348313
Filing Date: 2002-02-19
Classification: C12Q,Y10T

Abstract:
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).