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Double strand driver cDNA was prepared by low cycling PCR using phosphorylated driver3 primer and driver5 primer (TCCTAAGGTAGCGAGGCCATTACGGCCGGG) which is complementary to the 5'driver adaptor.
Double strand driver cDNA was synthesised using driver3 primer which is phosphorylated and driver5 primer (TCCTAAGGTAGCGAGGCCATTACGGCCGGG) which is complementary to the 5' driver adaptor.
The anti-sense strand of the double strand driver cDNA which is phosphorylated at the 5'end was destroyed by treating it with lambda exonuclease.
Tester DNA was then melted and reannealed with an excess of driver DNA and the remaining single strand driver DNA (normalized or subtracted library) was then purified by hydroxyapatite chromatography.
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