Method for identification of genetic markers
First Claim
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1. A method for identifying genetic markers comprising:
- a) obtaining genomic DNA from a first source having a first phenotype or trait of interest;
b) digesting said DNA with at least one restriction endonuclease having a degenerate recognition or cleavage sequence comprising at least one degenerate base said degenerate sequence represented by the formula Nm, where N is the extent of degeneracy, and m is the number of degenerate bases, to produce restriction fragments having Nm different single-stranded overhangs for each restriction endonuclease;
c) ligating restriction fragments having identical overhangs with a series of adapters whose sequences are complementary to said overhangs;
d) amplifying said restriction fragments;
e) separating said amplified restriction fragments to produce a restriction fragment pattern;
f) obtaining genomic DNA from at least one additional source having a second phenotype or trait of interest;
g) repeating b) through e) for said DNA from said at least one additional source;
h) comparing the restriction fragment patterns from each source;
i) selecting restriction fragments associated with a phenotype or trait of interest; and
j) using said selected restriction fragments as markers for said phenotype or trait of interest.
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Abstract
Methods are provided for identifying genetic markers and for determining methylation patterns using restriction endonucleases having degenerate recognition or cleavage sequences.
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Citations
24 Claims
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1. A method for identifying genetic markers comprising:
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a) obtaining genomic DNA from a first source having a first phenotype or trait of interest;
b) digesting said DNA with at least one restriction endonuclease having a degenerate recognition or cleavage sequence comprising at least one degenerate base said degenerate sequence represented by the formula Nm, where N is the extent of degeneracy, and m is the number of degenerate bases, to produce restriction fragments having Nm different single-stranded overhangs for each restriction endonuclease;
c) ligating restriction fragments having identical overhangs with a series of adapters whose sequences are complementary to said overhangs;
d) amplifying said restriction fragments;
e) separating said amplified restriction fragments to produce a restriction fragment pattern;
f) obtaining genomic DNA from at least one additional source having a second phenotype or trait of interest;
g) repeating b) through e) for said DNA from said at least one additional source;
h) comparing the restriction fragment patterns from each source;
i) selecting restriction fragments associated with a phenotype or trait of interest; and
j) using said selected restriction fragments as markers for said phenotype or trait of interest. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for determining DNA methylation patterns comprising:
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a) obtaining genomic DNA from a first source;
b) digesting said DNA with at least one restriction endonuclease having a degenerate recognition or cleavage sequence comprising at least one degenerate base said degenerate sequence represented by the formula Nm, where N is the extent of degeneracy, and m is the number of degenerate bases, to produce restriction fragments having Nm different single-stranded overhangs for each restriction endonuclease;
c) further digesting said DNA with at least one additional restriction endonuclease that produces a single-stranded overhang, said at least one additional restriction endonuclease being a methylation sensitive endonuclease;
d) ligating restriction fragments having identical overhangs with a series of adapters whose sequences are complementary to said overhangs;
e) amplifying said restriction fragments;
f) separating said amplified restriction fragments to produce a methylation fragment pattern. - View Dependent Claims (24)
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Specification