Patent ID: 6737253
Filing Date: 2004-05-18
Classification: C12Q

Abstract:
A method of amplifying a nucleotide sequence of interest wherein the nucleotide sequence comprises at least one region of known sequence, wherein the region of known sequence comprises, in a 3â€² to 5â€² direction: a first known region and a second known region wherein the first and second known regions are immediately adjacent each other and wherein the method comprises:1) a first amplification step of at least two cycles comprising amplifying the nucleotide sequence of interest using at least (a) as a template, a sequence comprising at least the nucleotide sequence of interest; (b) one first primer having, in a 5â€² to 3â€² direction: a 5â€² tag sequence; a degenerate sequence corresponding to the first known region; and a sequence complementary to the second known region of the nucleotide sequence of interest; and (c) a reverse primer, deoxyribonucleoside triphosphates (dNTPs) and suitable enzyme conducted under reaction conditions such that the first amplification step generates a first amplification product; and 2) a second amplification step comprising amplifying the nucleotide sequence of interest using at least (a) as a template, the first amplification product; and (b) one second primer having, in a 5â€² to 3â€² direction: a sequence which hybridises to the complementary strand of the 5â€² tag sequence of the first primer and a sequence complementary to the first known region of the nucleotide sequence of interest; which second amplification step generates a second amplification product.