Patent ID: 6066457
Filing Date: 2000-05-23
Classification: C12N,C12Q

Abstract:
A method of processing nucleic acid starting material composed of a collection of single stranded nucleic acid molecules so as to produce a mixture of shorter directional random length DNA fragments suitable for enabling substantially uniform global PCR amplification to be achieved yielding an amplified product representative of the total original single stranded nucleic acid, said method comprising the steps of:(a) deriving from said nucleic acid starting material an immobilised set of first strand cDNA molecules each anchored at one end to a first solid phase support;(b) carrying out a first random priming operation using said set of anchored first strand cDNA molecules as templates to synthesise a first set of random length single strand DNA fragments each incorporating at one end a first known nucleotide sequence and a terminal anchor group adapted to bind to complementary receptor material;(c) separating said first set of random length single strand DNA fragments from the set of anchored first strand cDNA molecules;(d) carrying out a second random priming operation using said first set of random length single strand DNA fragments as templates to synthesise a second set of single strand DNA fragments that each incorporate a second known nucleotide sequence at the opposite end to said first known nucleotide sequence;(e) contacting the reaction mixture from (d) with a second solid phase support carrying receptor material complementary to said terminal anchor groups of said first set of random length single strand DNA fragments whereby said first set of random length single strand DNA fragments are anchored and immobilized on said second solid phase support;(f) separating said newly synthesized second set of single strand DNA fragments from the immobilised first set of random length DNA fragments, said second set of single strand DNA fragments then providing a desired mixture of shorter random length single stranded DNA molecules suitable for subjecting to PCR amplification.