Methods for producing probes capable of distinguishing DNA from related sources
First Claim
1. A method for producing probes capable of distinguishing at least one sequence difference between DNA from two different eukaryotic sources, said method comprising:
- completely digesting separately the DNA from said two different sources with a restriction endonuclease to provide digested fragments, wherein one of said sources is driver DNA, and the other source is tester DNA, wherein said tester DNA comprises target DNA, wherein said target DNA comprises sequence differences between the DNA of said two sources;
ligating a first set of adaptors to said digested fragments and amplifying said fragments using primers to one of the strands of said first set adaptors to provide amplified amounts of fragments of said digested fragments of less than about 2 kbp as amplicons;
carrying out a first round of the following steps for enrichment of target DNA;
removing said first set of adaptors from said amplicons and ligating a second set of adaptors to the 5'"'"' ends of the amplicons of tester DNA;
combining under melting and annealing conditions said tester amplicons with a large excess of at least about 5-fold of driver amplicons, whereby a portion of the resulting dsDNA comprises self-annealed tester DNA including target DNA;
filling in the 3'"'"' ends of annealed DNA;
amplifying said portion of said dsDNA with primers complementary to one of said strands of said second set of adaptors to enrich for target DNA;
optionally repeating said first round of steps as a second round or successive round, to provide DNA sequences which serve to identify differences in DNA sequences between said tester source and said driver source.
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Abstract
Methodology is provided for developing probes for identifying sequence differences between two related DNA populations, sets of DNA fragments or collections of restriction-endonuclease-cleaved DNA or cDNA. The method employs an initial stage to obtain a representation of both DNA populations, namely using the PCR to produce relatively short fragments, referred to as amplicons. Tester amplicons containing target DNA, sequences of interest, are ligated to adaptors and mixed with excess driver amplicons under melting and annealing conditions, followed by PCR amplification. The process may be repeated so as to greatly enrich the target DNA. Optionally, the target DNA may then be cloned and the DNA used as probes.
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Citations
14 Claims
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1. A method for producing probes capable of distinguishing at least one sequence difference between DNA from two different eukaryotic sources, said method comprising:
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completely digesting separately the DNA from said two different sources with a restriction endonuclease to provide digested fragments, wherein one of said sources is driver DNA, and the other source is tester DNA, wherein said tester DNA comprises target DNA, wherein said target DNA comprises sequence differences between the DNA of said two sources; ligating a first set of adaptors to said digested fragments and amplifying said fragments using primers to one of the strands of said first set adaptors to provide amplified amounts of fragments of said digested fragments of less than about 2 kbp as amplicons; carrying out a first round of the following steps for enrichment of target DNA; removing said first set of adaptors from said amplicons and ligating a second set of adaptors to the 5'"'"' ends of the amplicons of tester DNA; combining under melting and annealing conditions said tester amplicons with a large excess of at least about 5-fold of driver amplicons, whereby a portion of the resulting dsDNA comprises self-annealed tester DNA including target DNA; filling in the 3'"'"' ends of annealed DNA; amplifying said portion of said dsDNA with primers complementary to one of said strands of said second set of adaptors to enrich for target DNA; optionally repeating said first round of steps as a second round or successive round, to provide DNA sequences which serve to identify differences in DNA sequences between said tester source and said driver source. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method for producing probes capable of distinguishing at least one sequence difference between genomes from two cellular sources, said method comprising:
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completely digesting separately the DNA from said two different sources with a restriction endonuclease having a nucleotide recognition sequence of at least 4 nucleotides, wherein one of said sources is driver DNA, and the other source is tester DNA, wherein said tester DNA comprises target DNA, wherein said target DNA comprises sequence differences between the genomes of said two sources; ligating a first set of adaptors to said digested fragments and amplifying said fragments using primers to one of the strands of said first set adaptors to provide by means of the polymerase chain reaction amplified amounts of fragments of said digested fragments of less than about 2 kbp as amplicons; removing said first set of adaptors from said amplicons; carrying out a first round of the following steps for enrichment of target DNA; ligating a second set of adaptors to the 5'"'"' end of amplicons of tester DNA; combining under melting and annealing conditions said tester amplicons with a large excess of driver amplicons, whereby a portion of the resulting dsDNA comprises self-annealed tester DNA including target DNA; filling in 3'"'"' overhangs; amplifying said dsDNA with primers to one of said strands of said second set of adaptors to enrich for target DNA; optionally removing said second set of adaptors and ligating a new set of adaptors; repeating said first round of steps for at least a total of 2 rounds, using a different set of adaptors in each successive round for said 2 rounds to provide a DNA composition comprising a further enriched amount of target DNA; cloning said DNA composition to provide clones having a homogeneous probe of putative target DNA; with the proviso that when a plurality of probes of putative target DNA are obtained, optionally including the additional step of; hybridizing said probes of putative target DNA with driver and tester amplicons, whereby probes of putative target DNA binding to both driver and tester amplicons are discarded. - View Dependent Claims (10, 11, 12)
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13. A method for producing probes capable of distinguishing at least one sequence difference between genomes from a neoplastic cell source and a normal cell source of the same species, said method comprising:
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completely digesting separately the DNA from said two sources with a restriction endonuclease having a nucleotide recognition sequence of at least 4 nucleotides, wherein said normal cell source is driver DNA, and said neoplastic cell source is tester DNA, wherein said tester DNA comprises target DNA, wherein said target DNA comprises sequence differences between the genomes of said two sources comprising at least one of an insertion, deletion, rearrangement or DNA amplification defining target DNA; ligating a first set of adaptors to said digested fragments and amplifying by means of the polymerase chain reaction said fragments using primers to one of the strands of said first set of adaptors to provide amplified amounts of fragments of said digested fragments of less than about 2 kbp as amplicons; removing said first set of adaptors from said amplicons; carrying out a first round of the following steps for enrichment of target DNA; ligating a second set of adaptors to 5'"'"' ends of amplicons of tester DNA; combining under melting and annealing conditions said tester amplicons with a large excess of at least about 5 fold driver amplicons, whereby a portion of the resulting dsDNA comprises self-annealed tester DNA including target DNA; filling in the 3'"'"' ends of overhangs; amplifying said dsDNA with primers to one of said strands of said second set of adaptors to enrich for target DNA; repeating said first round of steps for at least 1 additional round, using a different set of adaptors as to the previous round in each successive round to provide a DNA composition comprising a further enriched amount of target DNA; and cloning said DNA composition to provide clones having a homogeneous probe of target DNA.
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14. A method for producing probes capable of distinguishing at least one sequence difference between genomes from a neoplastic cell source and a normal cell source of the same species, said method comprising:
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completely digesting separately the DNA from said two sources with a restriction endonuclease having a nucleotide recognition sequence of at least 4 nucleotides, wherein said neoplastic cell source is driver DNA, and said normal cell source is tester DNA, wherein said tester DNA comprises target DNA, wherein said target DNA comprises sequence differences between the genomes of said two sources comprising loss of heterozygosity, homozygosity or hemizygous loss to define target DNA; ligating a first set of adaptors to said digested fragments and amplifying by means of the polymerase chain reaction said fragments using primers to one of the strands of said first set of adaptors to provide amplified amounts of fragments of said digested fragments of less than about 2 kbp as amplicons; removing said first set of adaptors from said amplicons; carrying out a first round of the following steps for enrichment of target DNA;
ligating a second set of adaptors to 5'"'"' ends of amplicons of tester DNA;combining under melting and annealing conditions said tester amplicons with a large excess of at least about 5 fold of driver amplicons, whereby a portion of the resulting dsDNA comprises self-annealed tester DNA including target DNA; filling in the 3'"'"' ends of overhangs; amplifying said dsDNA with primers to one of said strands of said second set of adaptors to enrich for target DNA; repeating said first round of steps for at least 1 round, using a different set of adaptors as to the previous round in each successive round to provide a DNA composition comprising a further enriched amount of target DNA; and cloning said DNA composition to provide clones having a homogeneous probe of target DNA.
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Specification