[특허]Multiplexed systems for nucleic acid sequencing

Multiplexed systems for nucleic acid sequencing 원문보기

IPC분류정보
국가/구분	United States(US) Patent 등록
국제특허분류(IPC7판)	C12Q-001/68
출원번호	US-0452157 (2003-05-30)
등록번호	US-7291460 (2007-11-06)
발명자 / 주소	Lafferty,Mike Tweedy,Charles Butler,James
출원인 / 주소	Verenium Corporation
인용정보	피인용 횟수 : 0 인용 특허 : 47

초록

Methods for the sequencing of polynucleotides are provided. Also provided are multiplex systems for sequencing polynucleotides using the disclosed methods, kits for use with the disclosed methods and methods for diagnosing diseases and adverse drug reactions using the disclosed methods.

대표청구항 ▼

What is claimed is: 1. A method for determining the sequence of a polynucleotide comprising, a) providing at least one population of polynucleotides wherein members of the population share a nucleotide sequence; b) providing 4x sets of sequencing adaptors, wherein each set of sequencing adaptors comprises one of 4x possible combinations of nucleotides in a single-stranded region of x nucleotides long, a nuclease recognition site for a nuclease that produces a single-stranded region of x nucleotides long, and a detectable optical marker that is unique to that set of sequencing adaptors, where x is an integer of 3 or more; c) attaching a first end of said polynucleotides to a substrate and ligating to a second end of said polynucleotides a set of initial adaptors comprising a recognition site for a nuclease; d) treating said polynucleotides with a nuclease specific for the recognition site of the initial adaptors, wherein said nuclease cleaves the polynucleotides but not the recognition site to produce digested polynucleotides comprising a single-stranded region of x nucleotides; e) simultaneously contacting the digested polynucleotides with the 4x sets of sequencing adaptors under conditions such that the sequencing adaptors hybridize to the digested polynucleotides with minimal mismatches; f) ligating the hybridized sequencing adaptors to the polynucleotides to produce adaptor-ligated polynucleotides; g) removing unhybridized sequencing adaptors; h) determining the identity of the hybridized sequencing adaptors by their unique marker thus simultaneously determining the identity and order of x nucleotides in said polynucleotides; i) treating the adaptor-ligated polynucleotides with a nuclease specific for the recognition site of the sequencing adaptors, wherein said nuclease cleaves the polynucleotides but not the recognition site to produce digested polynucleotides comprising a single-stranded region of x nucleotides and a sequencing adaptor fragment; and j) repeating e) through i) at least once, or until the sequence of the polynucleotide has been determined wherein said optical markers are selected using the formula: 2. A method for determining the sequence of a polynucleotide comprising, a) providing at least one population of polynucleotides wherein members of the population share a nucleotide sequence; b) providing 4x sets of sequencing adaptors, wherein each set of sequencing adaptors comprises one of 4x possible combinations of nucleotides in a single-stranded region of x nucleotides long, a nuclease recognition site for a type IIs restriction enzyme that produces a single-stranded region of x nucleotides long, and a fluorescent microsphere with an emission spectrum that is unique to that set of sequencing adaptors, where x is an integer of 3 or more and wherein said microspheres are selected using the formula: c) attaching a first end of said polynucleotides to a substrate and ligating to a second end of said polynucleotides a set of initial adaptors comprising a recognition site for a type IIs restriction enzyme; d) treating said polynucleotides with a type IIs restriction enzyme specific for the recognition site of the initial adaptors to produce digested polynucleotides comprising a single-stranded region of x nucleotides where x is equal to the number of nucleotides in the single-stranded region of the sequencing adaptors; e) simultaneously contacting the digested polynucleotides with the 4x sets of sequencing adaptors under stringent conditions such that the sequencing adaptors hybridize to the digested polynucleotides with minimal mismatches; f) ligating the hybridized sequencing adaptors to the polynucleotides to produce adaptor-ligated polynucleotides; g) removing unhybridized sequencing adaptors; h) determining the identity of the sequencing adaptors while hybridized to the polynucleotide to be sequenced by their fluorescent microsphere, thus simultaneously determining the identity and order of x nucleotides in said polynucleotides; i) treating the adaptor-ligated polynucleotides with a type IIs restriction enzyme specific for the recognition site of the sequencing adaptors to produce digested polynucleotides comprising a single-stranded region of x nucleotides and a sequencing adaptor fragment; j) removing the sequencing fragment; and k) repeating e) through j) at least once, or until the sequence of the polynucleotide has been determined.

이 특허에 인용된 특허 (47)

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Multiplexed systems for nucleic acid sequencing 원문보기

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Multiplexed systems for nucleic acid sequencing 원문보기

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