SYSTEM AND METHOD FOR NUCLEIC ACID SEQUENCING BY POLYMERASE SYNTHESIS

• US 20080241833A1
• Filed: 06/01/2007
• Published: 10/02/2008
• Est. Priority Date: 12/14/1998
• Status: Abandoned Application

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Williams, John G.K.
• Application Number:
  US11/757,289
• Publication Number:
  US 20080241833A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

Claims

1-143. -143. (canceled)

144. A method for genotyping a target nucleic acid, said method comprising:

providing a primer nucleic acid which complements a region of a target nucleic acid to form a hybridized complement;

immobilizing said target nucleic acid complement to a solid support by means that prevents release;

contacting said target nucleic acid complement with a least one type of labeled nucleotide triphosphate (NTP), wherein each type of NTP is differently labeled; and

detecting the incorporation of said labeled NTP into a single molecule of the primer, to genotype said target nucleic acid.

145. The method of claim 144, wherein said target nucleic acid complement is attached to said solid support by a bonding selected from the group consisting of covalent bonding, ionic boding and hydrophobic bonding.

146. The method of claim 145, wherein said target nucleic acid is attached to the solid support by covalent bonding.

147. The method of claim 144, wherein said target nucleic acid comprises a single nucleotide polymorphism.

148. The method of claim 144, wherein each type of NTP is differently labeled on the γ

-phosphate.

149. The method of claim 144, wherein said primer length is at least 10 bp in length.

150. The method of claim 144, wherein said primer length is at least between 15 and 30 bp in length.

151. The method of claim 144, wherein said primer binds to synthetic oligonucleotide adaptors joined to the target nucleic acid.

152. The method of claim 144, wherein said primer is designed to hybridize to a known internal site on the target nucleic acid.