Methods for reducing the complexity of DNA sequences
First Claim
1. 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.
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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.
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Citations
25 Claims
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1. A method for reducing the complexity of DNA sequences in a DNA sample by generating non-overlapping subsets of sequences which comprises:
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(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.- View Dependent Claims (2, 3, 4, 5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
(i) (a) blocking the duplex ends with a modified nucleotide resistant to exonuclease activity;
(b) nicking the duplexes obtained in step (b) with MutSLH;
(c) digesting the nicked duplexes with an exonuclease; and
(d) isolating the single-stranded DNA generated by the exonuclease;
or(ii) (e) blocking the 3′
ends of the fragments with terminal transferase and dideoxynucleoside triphosphates;
(f) treating the blocked fragments with MutSLH to obtain nicked fragments;
(g) attaching a tag to the nicks with a terminal transferase;
(h) trapping the tagged fragments with a capture agent to obtain subsets;
(i) melting off the complementary strands; and
then(iii) (j) amplifying the single stranded DNA obtained in either (i) or (ii) with a polymerase chain reaction or replicating the DNA in a single primer extension reaction;
or(k) employing adapters containing a specific sequence of an RNA polymerase promoter and selectively transcribing the remaining duplex DNA with the corresponding RNA polymerase and directly probing with the RNA or reconverting back into double-stranded cDNA;
ordenaturing the duplexes, diluting them, annealing them to an excess of a splint oligonucleotide containing sequences complementary to both arms of the Y-adapters or conventional adapters attached to the 3′
-end of the unmethylated DNA fragments, ligating the single-stranded DNA sufficiently diluted such that the ligation of the ends of a DNA strand will occur only when they are held together by the splint oligonucleotide, and then amplifying the resulting circular DNA using the rolling circle method, using the splint oligonucleotide or an oligonucleotide complementary to an adapter which has been elongated with a DNA polymerase capable of strand displacement.
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17. A method according to claim 16 wherein single-stranded DNA is either partially or completely removed after digesting with an exonuclease.
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18. A method according to claim 17 wherein the step is carried out with a benzoylated naphthoylated DEAE-cellulose column.
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19. A method according to claim 16 wherein products obtained in steps (i) or (ii) are amplified in step (iii) by transcription using a T7 RNA polymerase promoter as an adapter.
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20. A method for selectively isolating mismatched or perfectly matched DNA from the subsets obtained in claim 16 comprising either:
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(i) (a) isolating the single-stranded DNA generated from mismatched DNA by the exonuclease; and
(b) amplifying the single stranded DNA with a polymerase chain reaction using one primer complementary to the 3′
-hydroxyl-ended arm of the methylated DNA strand and another primer containing sequences from the 5′
-phosphate terminated arm of the methylated DNA strand or replicating the DNA in a single primer extension reaction;
or(ii) (c) attaching a tag to the nicks with terminal transferase;
(d) trapping the tagged fragments with a capture agent to obtain subsets;
(e) obtaining complementary strands by melting them off; and
then either(f) carrying out step (b);
or(iii) (g) extending the nick that MutSLH created in mismatched DNA duplexes using a DNA polymerase;
(h) amplifying the extended fragment by PCR using one primer complementary to the appropriate arm of the extended fragment and another primer containing a sequence from the appropriate arm of the Y-adapter on the extended fragment;
or(i) capturing perfectly matched DNA by denaturing the duplexes, diluting them and then annealing them to an excess of a splint oligonucleotide containing sequences complementary to one arm of the Y-adapter attached to the 3′
-end of the unmethylated DNA fragments, and adjacent sequences complementary to the arm of the Y-adapter that had been attached to the 5′
-end of the unmethylated DNA strand in heteroduplexes; and
(j) ligating the single-stranded DNA diluted sufficiently such that the ligation of the ends of a DNA strand will occur only when they are held together by the splint oligonucleotide; and
then(k) using either the splint oligonucleotide or a second oligonucleotide complementary to the single-stranded circle to amplify the circularized DNA by the rolling circle method.
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21. A method according to claim 20 wherein the tag employed in (ii) is biotin and the capture agent is avidin or strepavidin.
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22. A method according to claim 20 wherein the rolling circle amplification in (iii) is accomplished using a reconstituted bacterial polymerase III holoenzyme system that includes clamp proteins, and clamp loading proteins.
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23. A method according to claim 22 wherein the holoenzyme system further comprises a helicase.
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24. A method according to claim 16 wherein amplification using a polymerase chain reaction is employed.
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25. A method according to claim 16 wherein the tag employed in (ii) is biotin and the capture agent is avidin or strepavidin.
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6. A method for reducing the complexity of DNA sequences in a DNA sample by generating non-overlapping subsets of sequences which comprises:
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(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.- View Dependent Claims (7, 8)
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9. A method for reducing the complexity of DNA sequences in a DNA sample by generating non-overlapping subsets of sequences which comprises:
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(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. - View Dependent Claims (10)
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Specification