Multiplexed systems for nucleic acid sequencing

• US 7,291,460 B2
• Filed: 05/30/2003
• Issued: 11/06/2007
• Est. Priority Date: 05/31/2002
• Status: Expired due to Fees

• Current Assignee: BP Corporation North America Incorporated (BP PLC)
• Original Assignee: Verenium Corporation (BASF SE)
• Inventors: Butler, James, Tweedy, Charles, Lafferty, Mike
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US10/452,157
• Publication Number:
  US 20030224439A1
• Time in Patent Office: 1,621 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

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.

Claims

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 4^x sets of sequencing adaptors, wherein each set of sequencing adaptors comprises one of 4^x 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 4^x 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 4^x sets of sequencing adaptors, wherein each set of sequencing adaptors comprises one of 4^x 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;