Selective restriction fragment amplification: fingerprinting
First Claim
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1. Process for amplification of at least one restriction fragment from a target DNA regardless of whether its nucleotide sequence is unknown, which process comprises:
- (a) digesting said target DNA with at least one specific restriction endonuclease to fragment it into restriction fragments;
(b) ligating to the restriction fragments obtained from the target DNA at least one double-stranded synthetic oligonucleotide adaptor having one end which is compatible to be ligated to one or both of the ends of the restriction fragments to thereby produce tagged restriction fragments of the target DNA;
(c) contacting said tagged restriction fragments under hybridizing conditions with at least one oligonucleotide primer;
(d) wherein said at least one oligonucleotide primer is structurally complementary to at least part of said at least one double stranded oligonucleotide adaptor and to at least part of the restriction site of said at least one specific restriction endonuclease(s) used in step (a), in the tagged restriction fragment, and wherein at least one oligonucleotide primer includes at its 3'"'"' end, a selected nucleotide sequence comprising one to 4 nucleotide residues located immediately adjacent to the restriction site for said at least one specific restriction endonuclease, said selected nucleotide sequence being structurally complementary to a sequence which is immediately adjacent to the restriction site in the target DNA of at least one tagged restriction fragment; and
(e) amplifying or elongating said tagged restriction fragments using said at least one oligonucleotide primer in the presence of the required nucleotides and DNA polymerase.
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Abstract
The invention relates to a process for the controlled amplification of at least one part of a starting DNA containing a plurality of restriction sites for a determined specific restriction endonuclease, and of which at least part of its nucleic acid is unknown.
Application of this process to human, animal or plant DNA fingerprinting, to identification of restriction fragment length polymorphisms.
Kit for the application of the process.
283 Citations
30 Claims
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1. Process for amplification of at least one restriction fragment from a target DNA regardless of whether its nucleotide sequence is unknown, which process comprises:
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(a) digesting said target DNA with at least one specific restriction endonuclease to fragment it into restriction fragments; (b) ligating to the restriction fragments obtained from the target DNA at least one double-stranded synthetic oligonucleotide adaptor having one end which is compatible to be ligated to one or both of the ends of the restriction fragments to thereby produce tagged restriction fragments of the target DNA; (c) contacting said tagged restriction fragments under hybridizing conditions with at least one oligonucleotide primer; (d) wherein said at least one oligonucleotide primer is structurally complementary to at least part of said at least one double stranded oligonucleotide adaptor and to at least part of the restriction site of said at least one specific restriction endonuclease(s) used in step (a), in the tagged restriction fragment, and wherein at least one oligonucleotide primer includes at its 3'"'"' end, a selected nucleotide sequence comprising one to 4 nucleotide residues located immediately adjacent to the restriction site for said at least one specific restriction endonuclease, said selected nucleotide sequence being structurally complementary to a sequence which is immediately adjacent to the restriction site in the target DNA of at least one tagged restriction fragment; and (e) amplifying or elongating said tagged restriction fragments using said at least one oligonucleotide primer in the presence of the required nucleotides and DNA polymerase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18)
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11. A process for amplification of at least one restriction fragment from a target DNA regardless of whether its nucleotide sequence is unknown, which process comprises:
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(a) digesting said target DNA with at least one specific restriction endonuclease to fragment it into restriction fragments; (b) ligating to the restriction fragments obtained from the target DNA at least one double-stranded synthetic oligonucleotide adaptor having one end which is compatible to be ligated to one or both of the ends of the restriction fragments to thereby produce tagged restriction fragments of the target DNA; (c) contacting said tagged restriction fragments under hybridizing conditions with at least one oligonucleotide primer; (d) wherein said at least one oligonucleotide primer is structurally complementary to at least part of said at least one double stranded oligonucleotide adaptor and to at least part of the restriction site of said at least one specific restriction endonuclease(s) used in step (a), in the tagged restriction fragment, and wherein at least one oligonucleotide primer includes at its 3'"'"' end, a selected nucleotide sequence comprising one to 4 nucleotide residues located immediately adjacent to the restriction site for said at least one specific restriction endonuclease, said selected nucleotide sequence having structurally complementary to a sequence which is immediately adjacent to the restriction site in the target DNA of at least one tagged restriction fragment; (e) amplifying or elongating said tagged restriction fragments using said at least one oligonucleotide primer in the presence of the required nucleotides and DNA polymerase, (f) isolating at least one amplified or elongated DNA fragment; (g) determining the nucleotide sequence of the first 8-10 nucleotide residues internally adjacent to the restriction sites at both ends of said at least one amplified or elongated DNA fragment; (h) designing oligonucleotide primers having a nucleotide sequence according to the at least one oligonucleotide primer of step (d) wherein the selected nucleotide sequence comprises nucleotide residues which correspond to the first 8-10 nucleotide residues internally adjacent to the restriction sites at both ends of said DNA fragment. - View Dependent Claims (12)
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13. A process for identifying polymorphisms between different target DNAs originating from the same species comprising amplifying at least one restriction fragment from said different target DNAs regardless of whether their nucleotide sequences are unknown, which process comprises:
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(a) digesting said target DNAs with at lest one specific restriction endonuclease to fragment them into restriction fragments; (b) ligating to the restriction fragments obtained from the target DNAs at least one double-stranded synthetic oligonucleotide adaptor having one end which is compatible to be ligated to one or both of the ends of the restriction fragments to thereby produce tagged restriction fragments of the target DNAs; (c) contacting said tagged restriction fragments under hybridizing conditions with at least one oligonucleotide primer; (d) wherein said at least one oligonucleotide primer is structurally complementary to at least part of said at least one double stranded oligonucleotide adaptor and to at least part of the restriction site of said at least one specific restriction endonuclease(s) used in step (a), in the tagged restriction fragment, and wherein at least one oligonucleotide primer includes at its 3'"'"' end, a selected nucleotide sequence comprising one to 4 nucleotide residues located immediately adjacent to the restriction site for said at least one specific restriction endonuclease, said selected nucleotide sequence being structurally complementary to a sequence which is immediately adjacent to the restriction site in the target DNA of at least one tagged restriction fragment; (e) amplifying or elongating said tagged restriction fragments using said at least one oligonucleotide primer in the presence of the required nucleotides and DNA polymerase, (f) identifying or recovering the amplified or elongated tagged restriction fragments as produced in step (e) as DNA fingerprints, and (g) comparing the DNA fingerprints obtained from each of said target DNAs and identifying differences between the DNA fingerprints of the different target DNAs. - View Dependent Claims (19, 20, 21, 22, 27, 28, 29, 30)
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14. Process for identifying or recovering at least one amplified restriction fragment from a target DNA regardless of whether its nucleotide sequence is unknown, which process comprises:
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(a) digesting said target DNA with at least one specific restriction endonuclease to fragment it into restriction fragments; (b) ligating to the restriction fragments obtained from the target DNA at least one double-stranded synthetic oligonucleotide adaptor having one end which is compatible to be ligated to one or both of the ends of the restriction fragments to thereby produce tagged restriction fragments of the target DNA; (c) contacting said tagged restriction fragments under hybridizing conditions with at least one oligonucleotide primer; (d) wherein said at least one oligonucleotide primer is structurally complementary to at least part of said at least one double stranded oligonucleotide adaptor and to at least part of the restriction site of said at least one specific restriction endonuclease(s) used in step (a), in the tagged restriction fragment, and wherein at least one oligonucleotide primer includes at its 3'"'"' end, a selected nucleotide sequence comprising one to 4 nucleotide residues located immediately adjacent to the restriction site for said at least one specific restriction endonuclease, said selected nucleotide sequence being structurally complementary to a sequence which is immediately adjacent to the restriction site in the target DNA of at least one tagged restriction fragment; (e) amplifying or elongating said tagged restriction fragments using said at least one oligonucleotide primer in the presence of the required nucleotides and DNA polymerase, and (f) identifying or recovering the amplified or elongated tagged restriction fragments as produced in step (e). - View Dependent Claims (23, 24, 25, 26)
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Specification