Methods and compositions for reducing the complexity of a nucleic acid sample
First Claim
1. A method of producing one or more polynucleotide samples having reduced complexity from a parent polynucleotide sample, said method comprising:
- (i) fragmenting polynucleotides in a parent polynucleotide sample;
(ii) ligating a first oligonucleotide adapter to said polynucleotide fragments;
(iii) annealing a first nucleic acid synthesis primer to said oligonucleotide adapter-ligated fragments, wherein said nucleic acid synthesis primer anneals such that its 3′
base is immediately upstream of a first differentiating nucleotide position in each fragment;
(iv) contacting said synthesis-primer annealed fragments with a differentiating nucleotide mix under nucleic acid synthesis conditions, wherein said differentiating nucleotide mix comprises one or more nucleotide that is differentially incorporated into said synthesis-primer annealed fragments according to the identity of the nucleotide at said first differentiating nucleotide position; and
(v) isolating polynucleotide fragments having a nucleotide of predetermined identity at said first differentiating nucleotide position, wherein said fragments are isolated based on the differential incorporation of said one or more nucleotide, thereby producing one or more polynucleotide samples having reduced complexity from said parent.
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Abstract
Aspects of the present invention are drawn to methods and compositions for sorting nucleic acid molecules into physically separate compartments according to the identity of a nucleotide base or sequence of bases at a specific location, resulting in the production of reduced complexity samples that find use in any number of downstream genetic analyses. Aspects of the methods of the invention include fragmenting a nucleic acid sample, e.g., with a restriction enzyme, ligating an adaptor (or adaptors), and sorting the fragments based on the identity of the nucleotide base(s) positioned adjacent to the fragmentation site (e.g., the restriction enzyme cut site/or recognition site). Each round of sorting produces binned samples having reduced complexity over the parent sample.
23 Citations
19 Claims
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1. A method of producing one or more polynucleotide samples having reduced complexity from a parent polynucleotide sample, said method comprising:
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(i) fragmenting polynucleotides in a parent polynucleotide sample; (ii) ligating a first oligonucleotide adapter to said polynucleotide fragments; (iii) annealing a first nucleic acid synthesis primer to said oligonucleotide adapter-ligated fragments, wherein said nucleic acid synthesis primer anneals such that its 3′
base is immediately upstream of a first differentiating nucleotide position in each fragment;(iv) contacting said synthesis-primer annealed fragments with a differentiating nucleotide mix under nucleic acid synthesis conditions, wherein said differentiating nucleotide mix comprises one or more nucleotide that is differentially incorporated into said synthesis-primer annealed fragments according to the identity of the nucleotide at said first differentiating nucleotide position; and (v) isolating polynucleotide fragments having a nucleotide of predetermined identity at said first differentiating nucleotide position, wherein said fragments are isolated based on the differential incorporation of said one or more nucleotide, thereby producing one or more polynucleotide samples having reduced complexity from said parent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification