Patent ID: 6455255
Filing Date: 2002-09-24
Classification: C12Q

Abstract:
A method for performing subtractive hybridization using a tester sample and a driver sample to determine the presence of a nucleic acid sequence difference in the tester sample comprising:(a) separately isolating total nucleic acid from the tester sample and the driver sample, and generating double-stranded cDNA or DNA from said total nucleic acid from said tester sample and said driver sample; (b) digesting said double-stranded cDNA or DNA generated from the tester sample and the drive sample of step (a) with a restriction endonuclease in order to produce a set of restriction fragments for each sample; (c) ligating said driver and tester restriction fragments of each set of step (b) to a first oligonucleotide adapter set, and amplifying the resulting products with selective primers such that a subset of said restriction fragments of step (b) is amplified; (d) removing said selective primer sequences by restriction endonuclease digestion in order to produce tester and driver amplicons, ligating the 5â€²-ends of said driver and tester amplicons to a second oligonucleotide adapter set to form driver-control and tester, mixing the driver-control and tester separately with an excess of non-ligated driver amplicon each, denaturing said resulting mixtures, and allowing the denatured nucleic acid strands within each mixture to hybridize; (e) filling in the 3â€²-ends of the reannealed driver/tester and the reannealed driver/driver control using a thermostable DNA polymerase and amplifying the resulting sequences; (f) removing the remaining single-stranded DNA by digesting said DNA with a single-stranded DNA nuclease; (g) amplifying double-stranded DNA remaining after nuclease digestion; and (h) cleaving the subtraction products of the driver/tester and driver/driver-control with a restriction endonuclease to remove oligonucleotide adapters, and repeating steps (c) through (h), whereas steps (c) through (h) utilize an oligonucleotide adapter set not used in any previous round, wherein one round consists of performance of steps (c) through (h), and utilize as driver, for each new round, the restriction endonuclease-cleaved product of the driver/driver-control subtraction from immediately proceeding steps (c) through (h).