Methods of nucleic acid amplification and sequencing

• US 20080160580A1
• Filed: 08/17/2006
• Published: 07/03/2008
• Est. Priority Date: 09/30/1998
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Solexa Incorporated (Illumina Incorporated)
• Inventors: Mayer, Pascal, Turcatti, Gerardo, Kawashima, Eric, Adessi, Celine, Mermod, Jean-Jacques
• Granted Patent: US 8,652,810 B2
• Time in Patent Office: Days
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Methods for amplification and sequencing of at least one nucleic acid comprising the following steps: (1) forming at least one nucleic acid template comprising the nucleic acid(s) to be amplified or sequenced, wherein said nucleic acid(s) contains at the 5′ end an oligonucleotide sequence Y and at the 3′ end an oligonucleotide sequence Z and, in addition, the nucleic acid(s) carry at the 5′ end a means for attaching the nucleic acid(s) to a solid support; (2) mixing said nucleic acid template(s) with one or more colony primers X, which can hybridize to the oligonucleotide sequence Z and carries at the 5′ end a means for attaching the colony primers to a solid support, in the presence of a solid support so that the 5′ ends of both the nucleic acid template and the colony primers bind to the solid support; (3) performing one or more nucleic acid amplification reactions on the bound template(s), so that nucleic acid colonies are generated and optionally, performing at least one step of sequence determination of one or more of the nucleic acid colonies generated. Solid supports, kits and apparatus for use in these methods.

Claims

1-34. -34. (canceled)

35. A method for parallel amplification of a collection of single nucleic acid molecules of different sequence comprising:

a) treating the collection of nucleic acid molecules to be amplified such that each nucleic acid molecule to be amplified contains a known, common sequence at the 5′ and

3′

ends,

b) separating the collection of different nucleic molecules to be amplified such that individual molecules with different sequences are separated from each other, and

c) performing a single amplification reaction using amplification primers complementary to the known common sequences that simultaneously amplifies the whole collection of single nucleic acid molecules.

36. The method according to claim 35, wherein at least one amplification primer is immobilised on a solid support.

37. The method according to claim 36, wherein said solid support is a planar surface.

38. The method according to claim 36, wherein said solid support is a bead or microparticle.