Patent ID: 6673541
Filing Date: 2004-01-06
Classification: C12Q

Abstract:
A method for the amplification of DNA comprising:(a) providing a sample comprising DNA; (b) digesting the DNA to be amplified with a restriction endonuclease under conditions suitable to obtain DNA fragments of similar length, wherein said restriction endonuclease is capable of providing 5â€² overhangs wherein the terminal nucleotide of the overhang is phosphorylated or 3â€² overhangs wherein the terminal nucleotide of the overhang is hydroxylated on said DNA fragments; (c) annealing at least one primer to said DNA fragments and further performing steps selected from the group consisting of (i) hybridizing, simultaneously or subsequently, oligonucleotides representing a first primer to said 5â€² overhangs on said DNA fragments of step (b), and further hybridizing oligonucleotides representing a second primer to 3â€² overhangs generated by said first primer, wherein said first and second primer are of different lengths; ligating said second primer to said 5â€² overhangs; and removing said first primer from said DNA fragments; (ii) hybridizing, simultaneously or subsequently, oligonucleotides representing a first primer, wherein the nucleotide at the 5â€² terminus is phosphorylated, to said 5â€² overhangs on said DNA fragments of step (b), and further hybridizing oligonucleotides representing a second primer with said first primer; and ligating said first and second primer to said DNA fragments; and (iii) hybridizing oligonucleotides representing said primer to said 3â€² overhangs so that 5â€² overhangs are generated; and ligating said primer to recessed 5â€² ends of said DNA fragments; (d) filling in generated 5â€² overhangs; and (e) amplifying said DNA fragments with primers which are capable of hybridizing with the complementary strand of said primer(s) of step (c) to obtain said amplified DNA.