Patent ID: 6410276
Filing Date: 2002-06-25
Classification: C12N,C12Q,G01N

Abstract:
A method for increasing the number of copies of a target sequence, or the complement of said target sequence, in a polynucleotide sequence having a 3â€² region and a 5â€² region, and said target sequence therebetween, comprising the steps of:(1) preparing a first double-stranded DNA comprising a first promoter sequence upstream from said target sequence by: (a) contacting an RNA polynucleotide sequence comprising a target sequence with a first primer able to bind to a region upstream of said target sequence; (b) extending said first primer with a DNA polymerase to form a primer extension product of said first primer having a sequence complementary to said target sequence, (c) making said primer extension product of said first primer available for hybridization with a second primer, (d) hybridizing said primer extension product of said first primer with a second primer able to bind to said primer extension product of said first primer at a region upstream of the sequence complementary to said target sequence, wherein one of said first and second primers comprises a 5â€² promoter sequence, (e) extending said second primer, and if necessary said primer extension product of said first primer, with a DNA polymerase to prepare said first double-stranded DNA comprising a first promoter, and (2) transcribing multiple RNA copies from said first double-stranded DNA using a DNA-dependent RNA polymerase able to bind said promoter to thereby increase the number of copies of said target sequence, or the complement of said target sequence.