Making nucleic acid sequences in parallel and use

• US 7,544,793 B2
• Filed: 03/01/2006
• Issued: 06/09/2009
• Est. Priority Date: 03/10/2005
• Status: Active Grant

• Current Assignee: LC Sciences LC
• Original Assignee: Nuing Sheng, Qi Zhu, Xialoian Gao, Xiaochuan Zhou, Xiaolin Zhang
• Inventors: Gao, Xialoian, Sheng, Nuing, Zhou, Xiaochuan, Zhu, Qi, Zhang, Xiaolin
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Baughman; Molly E
• Application Number:
  US11/365,980
• Publication Number:
  US 20070031942A1
• Time in Patent Office: 1,196 Days
• Field of Search: None
• US Class Current: 536/25.3
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The present invention relates generally to the fields of genomics, synthetic biology and genetic engineering. More particularly, the present invention concerns the methods that enable parallel multiplex ligation and amplification on surface for making assemblies of nucleic acids of various biological applications and for analysis of biological samples such as DNA, RNA, and proteins.

Claims

1. A method for simultaneously producing two full-length duplex nucleic acid polymers which have identical sequences on a solid surface comprising multiple sites wherein each of the two full-length duplex nucleic acid polymers is located on a different site on the solid surface and wherein each duplex consists of a first strand and a second complementary strand each strand having a 5′

end and a 3′

end comprising;

(a) placing a first capture probe of a defined length which is identical to a portion of the 3′

end of the first strand on a first site on the solid surface;

(b) placing a second capture probe of a defined length which is identical to a portion of the 3′

end of the second strand on a second site on the solid surface;

(c) contacting the solid surface with a pool of partially double-stranded oligonucleotides, which comprise overlapping fragments of the full length duplex nucleic acid polymers to be produced;

(d) hybridizing the first and second capture probes with the pool of partially double-stranded oligonucleotides such that first and second full-length duplex nucleic acid polymers are formed in which there are nicks and gaps in the duplexes;

(e) washing the first and second full-length duplex nucleic acid polymers under conditions to remove nucleic acid polymers which do not have sequences which are identical to the two full-length duplex nucleic acid polymers; and

(f) ligating and extending the first gapped and unligated full-length duplex nucleic acid and the second gapped and unligated full-length duplex nucleic acid to produce two full-length duplex nucleic acid polymers which have identical sequences wherein the first full-length duplex nucleic acid polymer is attached to the first site through the 3′

end of the first strand and the second full-length duplex nucleic acid polymer is attached to the second site through the 3′

end of the second strand.

2. The method of claim 1 wherein the placing of the first and second capture probes is accomplished by synthesizing the probes de nova on the solid surface.

3. The method of claim 1 wherein the first capture probe has a length of between about 6 to about 100 nucleotides and the second capture probe has a length of between about 6 to about 100 nucleotides.