Iterative and regenerative DNA sequencing method
First Claim
1. A sequencing method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:
- a) digesting said double stranded nucleic acid segment with a restriction enzyme whose cleavage site is separate from its recognition site to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site;
b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain and a sequence identification region;
c) hybridizing said adaptor to said double stranded nucleic acid having said single-stranded overhang sequence to form a ligated molecule;
d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag, restriction enzyme recognition domain, and a portion of said sequence identification region of said adaptor;
e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and
f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.
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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.
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Citations
32 Claims
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1. A sequencing method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:
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a) digesting said double stranded nucleic acid segment with a restriction enzyme whose cleavage site is separate from its recognition site to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site;
b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain and a sequence identification region;
c) hybridizing said adaptor to said double stranded nucleic acid having said single-stranded overhang sequence to form a ligated molecule;
d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag, restriction enzyme recognition domain, and a portion of said sequence identification region of said adaptor;
e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and
f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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2. A sequencing method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:
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a) digesting said double stranded nucleic acid segment with a restriction enzyme whose cleavage site is separate from its recognition site, resulting in a trimmed end in said double stranded molecule;
b) providing an adaptor having a cycle identification tag and a restriction enzyme recognition domain;
c) ligating said adaptor to the trimmed end of said double stranded nucleic acid to form a ligated molecule;
d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag and said restriction enzyme recognition domain of the adaptor, and for a nucleotide in said trimmed end in said double stranded molecule;
e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and
f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site. - View Dependent Claims (29, 30, 31, 32)
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Specification