Genome walking by selective amplification of nick-translate DNA library and amplification from complex mixtures of templates

• US 20050032104A1
• Filed: 08/02/2004
• Published: 02/10/2005
• Est. Priority Date: 05/02/2001
• Status: Abandoned Application

• Current Assignee: Emmanuel Kamberov, Irina Sleptsova, Vladimir L. Makarov
• Original Assignee: Emmanuel Kamberov, Irina Sleptsova, Vladimir L. Makarov
• Inventors: Makarov, Vladimir L., Kamberov, Emmanuel, Sleptsova, Irina
• Application Number:
  US10/909,516
• Publication Number:
  US 20050032104A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

Improved methods and reagents for chromosome walking of nucleic acid are discussed herein. A library of amplifiable nick translation molecules is generated, and a chromosome walk is initiated from a known sequence in the nucleic acid by producing at least one nick translate molecule, sequencing part of the nick translate molecule, and producing a second nick translate molecule by initiating the primer extension from the region of the obtained sequence of the prior nick translate molecule.

Claims

1-59. -59. (Canceled).

60. A library of consecutive overlapping series of nucleic acid sequences from a DNA sample, wherein the library is generated by the method of:

generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from a known nucleic acid sequence in the DNA sample;

determining at least a partial sequence from said first nick translation product; and

generating at least a second amplifiable nick translation product, wherein said nick translation of said second amplifiable nick translation product initiates from the partial sequence of said first nick translation product.

61. The method of claim 60, further comprising at least partially digesting DNA molecules of the DNA sample with a sequence-specific endonuclease to generate a plurality of DNA fragments.

64. A library of consecutive overlapping series of nucleic acid sequences from a DNA sample, wherein the library is generated by the method of:

obtaining a DNA sample comprising a DNA molecule having a region comprising a known nucleic acid sequence;

at least partially digesting the DNA molecule with a sequence-specific endonuclease to generate a plurality of DNA fragments, wherein at least one DNA fragment has a region comprising the known nucleic acid sequence;

attaching a first adaptor molecule to ends of the DNA fragments to provide a nick translation initiation site;

subjecting the first adaptor-bound DNA fragment to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity, wherein the nick translation initiates from the known nucleic acid sequence, to generate a first nick translation product;

attaching a second adaptor molecule to the first nick translate product;

determining at least a partial sequence from the first nick translation product; and

generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product.

66. The method of claim 64, wherein the first adaptor comprises a label and the first nick translation product is isolated by the label.

65. The method of claim 65, further comprising separating the cleaved DNA fragments.