Patent ID: 6045994
Filing Date: 2000-04-04
Classification: C12Q

Abstract:
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.