Methods for reducing the complexity of DNA sequences
AssigneeMolecular Staging, Inc.
InventorsSherman Weissman, Roger Lasken, Xinghua Pan
Life of this patent
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Abstract
Genomic or cDNA, or fragments and mixtures thereof, can be screened by generation of subsets and then subjecting the subsets to mismatch scanning procedures. Alternatively, DNA fragments can be generated by cutting with a restriction endonuclease that generates variable overhangs. For either of the above methods, Y-shaped adapters having a region of non-complementary single-stranded DNA at the end can be used. Heterohybrid DNA, containing one DNA strand derived from each of two different samples, or homohybrids, containing DNA strands from the same sample, can be selected. Adapters attached to the ends of the fragments are designed to allow the selective isolation of homohybrid or heterohybrid DNA.
Claims25
A method for reducing the complexity of DNA sequences in a DNA sample by generating non-overlapping subsets of sequences which comprises:
- (a) digesting the DNA sample with a restriction enzyme to obtain DNA fragments;
- (b) ligating Y-shaped adapters to both ends of the fragments to obtain fragment-plus-adapter constructs;
- (c) removing excess adapters that are not ligated to the fragments;
- (d) amplifying the fragment-plus-adapter constructs by carrying out a PCR reaction with a first DNA primer that is complementary to one strand of the Y-shaped adapters and that, at the 3′-end of the primer, overlaps the DNA fragment by at least one base, and a second PCR primer that is complementary to at least a portion of the adapter region of the 3′-end of the DNA product formed by elongation of the first primer and which also anneals to the adapter region, and, at the 3′-end of the primer, overlaps the DNA fragment sequence by at least one base;
- and
- (e) identifying DNA products obtained in step (d) from each primer set as non-overlapping subsets, the primer sets differing by the 3′-nucleotides overlapping the fragment DNA, wherein each subset is less complex than the DNA sample.
A method for reducing the complexity of DNA sequences in a DNA sample by generating non-overlapping subsets of sequences which comprises:
- (a) digesting the DNA sample with a restriction enzyme to obtain DNA fragments;
- (b) ligating Y-shaped adapters to both ends of the fragments to obtain fragment-plus-adapter constructs;
- (c) removing excess adapters that are not ligated to the fragments;
- (d) denaturing the fragment-plus-adapter constructs and carrying out a single round of DNA replication using a DNA primer that is complementary to one strand of the Y-shaped adapters and that, at the 3′-end of the primer, overlaps the DNA fragment by at least one base;
- (e) denaturing the product of the reaction in step (d) and carrying out another single round of DNA replication using a second primer that has a tag attached to the 5′-end and that is complementary to at least a portion of the adapter region of the 3′-end of the DNA product formed by elongation of the first primer and which also anneals to the adapter region, and, at the 3′-end of the primer, overlaps the DNA fragment sequence by at least one base;
- (f) trapping the DNA product of (e) with a capture agent that binds the tag;
- and
- (g) identifying DNA products obtained in step (f) from each primer set as non-overlapping subset, the primer sets differing by the 3′-nucleotides overlapping the fragment DNA, wherein each subset is less complex than the DNA sample.
A method for reducing the complexity of DNA sequences in a DNA sample by generating non-overlapping subsets of sequences which comprises:
- (a) digesting the DNA sample with a restriction endonuclease that generates variable overhangs for which some of the sequence positions in the overhangs at cut sites can be any of the four deoxynucleotides;
- (b) carrying out separate Y-shaped adapter fragment ligations in which there are two different Y-shaped adapters in each ligation which differ from each other in the sequences of their non-complementary tails and in which the sequence of the adapter overhangs to be ligated to the fragments are unique for each ligation reaction;
- (c) removing the free adapters;
- (d) carrying out PCR reactions with the fragment-adapter constructs from each ligation using the corresponding primer sequences required to amplify one of the DNA strands of the fragment-adapter construct, wherein products of each PCR reaction are less complex than the DNA sample.
Dependencies are inferred from the claim text.