Genome walking by selective amplification of nick-translate DNA library and amplification from complex mixtures of templates
OwnerVladimir Makarov, Emmanuel Kamberov +1inventors
InventorsVladimir Makarov, Emmanuel Kamberov, Irina Sleptsova
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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.
Claims6
1-59. (Canceled).
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.
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.
The method of claim 65, further comprising separating the cleaved DNA fragments.
Dependencies are inferred from the claim text.