Representational approach to DNA analysis
First Claim
1. A kit comprising at least two probes from two different eukaryotic sources comprising a driver DNA and a tester DNA prepared according to a method for producing probes capable of distinguishing at least one sequence difference between DNA from said 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 said driver DNA, and the other source is said 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 by means of the polymerase chain reaction 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;
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 to serve as probes.
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Accused Products
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 CODA. 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.
40 Citations
12 Claims
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1. A kit comprising at least two probes from two different eukaryotic sources comprising a driver DNA and a tester DNA prepared according to a method for producing probes capable of distinguishing at least one sequence difference between DNA from said 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 said driver DNA, and the other source is said 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 by means of the polymerase chain reaction 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; 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 to serve as probes. - View Dependent Claims (2)
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3. A method for identifying the presence of target DNA in different cell sources of members of a eukaryotic population, said method comprising:
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(a) producing a probe to a target DNA according to a method 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 by means of the polymerase chain reaction 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; 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 to serve as probes; and (b) screening cells from a third cell source with said probe, wherein said third cell source is different from said first and second cell sources and is from a member of said eukaryotic population. - View Dependent Claims (4, 5, 6, 7, 8)
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9. A method for identifying DNA for distinguishing different members of a eukaryotic population, said method comprising:
producing a probe to target DNA according to a method 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 by means of the polymerase chain reaction 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; 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 to serve as probes; hybridizing one of said probes to DNA from tester DNA to identify the DNA site comprising said target DNA; and hybridizing DNA from said site with driver DNA to identify tester DNA different from driver DNA to use as additional probes. - View Dependent Claims (10, 11, 12)
Specification