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

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.

1 - 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.    
     
     
         65 . The method of  claim 65 , further comprising separating the cleaved DNA fragments.  
     
     
         66 . The method of  claim 64 , wherein the first adaptor comprises a label and the first nick translation product is isolated by the label.