NUCLEIC ACID SAMPLE PREPARATION METHODS AND COMPOSITIONS

• US 20160051959A1
• Filed: 08/17/2015
• Published: 02/25/2016
• Est. Priority Date: 12/27/2010
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Eshoo, Mark, Picuri, John, Motley, Stanley
• Application Number:
  US14/828,109
• Publication Number:
  US 20160051959A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The present invention provides compositions and methods for preparing a nucleic acid library in a multi-purpose buffer (e.g., employing whole genome amplification), where nucleic acid purification is not required between or during steps. In certain embodiments, small amounts of starting nucleic acid (e.g., genomic DNA) are employed and the steps are accomplished in a single container. In some embodiments, the nucleic acid library is subjected to sequencing methodologies or rolling circle amplification.

Claims

1. A method of preparing a nucleic acid library in a multi-purpose buffer, comprising:

a) combining a nucleic acid sample with a multi-purpose buffer, wherein said multi-purpose buffer comprises dNTPs and primers;

b) contacting said multi-purpose buffer with a plurality of substantially purified enzymes, wherein said enzymes have polymerase activity, kinase activity, and phosphatase activity, and wherein said contacting is under conditions such that amplified nucleic acid is generated;

c) treating said multi-purpose buffer such that sheared amplified nucleic acid is generated;

d) treating said multi-purpose buffer to inactivate said polymerase activity; and

e) contacting said multi-purpose buffer with a ligase and nucleic acid adapters under conditions such that an adapter-linked nucleic acid library is generated;

wherein the above steps are completed in said multi-purpose buffer without nucleic acid purification.