Methods for removing primer sequences and blocking restriction endonuclease recognition domains
First Claim
1. A method for removing all or a part of a primer sequence from a primer extended product, comprising:
- a) providing a primer sequence comprising a methylated portion of a restriction endonuclease recognition domain, wherein recognition of said domain by a restriction endonuclease requires at least one methylated nucleotide;
b) performing template-directed primer extension using said primer and a nucleic acid segment to generate a primer extended product; and
c) digesting said primer extended product with a restriction endonuclease that recognizes the resulting double-stranded restriction endonuclease recognition domain comprised by said primer sequence in said primer extended product.
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Abstract
An iterative and regenerative method for sequencing DNA is described. This method sequences DNA in discrete intervals starting at one end of a double stranded DNA segment. This method overcomes problems inherent in other sequencing methods, including the need for gel resolution of DNA fragments and the generation of artifacts caused by single-stranded DNA secondary structures. A particular advantage of this invention is that it can create offset collections of DNA segments and sequence the segments in parallel to provide continuous sequence information over long intervals. This method is also suitable for automation and multiplex automation to sequence large sets of segments.
29 Citations
17 Claims
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1. A method for removing all or a part of a primer sequence from a primer extended product, comprising:
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a) providing a primer sequence comprising a methylated portion of a restriction endonuclease recognition domain, wherein recognition of said domain by a restriction endonuclease requires at least one methylated nucleotide;
b) performing template-directed primer extension using said primer and a nucleic acid segment to generate a primer extended product; and
c) digesting said primer extended product with a restriction endonuclease that recognizes the resulting double-stranded restriction endonuclease recognition domain comprised by said primer sequence in said primer extended product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for blocking a restriction endonuclease recognition domain in a primer extended product, comprising:
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a) providing a primer with at least one modified nucleotide, wherein said modified nucleotide blocks an enzyme recognition domain, and wherein said primer comprises at least a portion of said enzyme recognition domain sequence;
b) performing template-directed primer extension using said primer and a nucleic acid segment to generate a primer extended product; and
c) digesting said primer extended product with an enzyme that recognizes a double-stranded enzyme recognition domain in said primer extended product;
to thereby block cutting mediated by said restriction endonuclease recognition domain in said primer extended product.- View Dependent Claims (11, 12, 13, 14, 15, 16, 17)
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Specification