This was achieved by the following: Given the right circumstances, denatured, single-stranded DNAs attempt to reassociate to double-stranded DNAs. Due to the likelihood of the two matching single strands of a frequently-occurring cDNA coming together in this mixture of molecules, the frequently-occurring cDNAs reassociate much more quickly than rare cDNAs. The remaining single strands can be accumulated by a subsequent selection.
Two different normalization methods using the Eppendorf Thermomixer (1) are described in the following passage:
A 20 g sample of a PCR-amplified cDNA library was precipitated with ethanol, dissolved in 25 l hybridizing buffer, covered with a layer of oil, denatured for five minutes at 95C and incubated for 12 hours at 68C in the Eppendorf Thermomixer for rehybridization.
After rehybridization, three different nucleic acids were present:
1. Rehybridized ds-cDNAs from highly expressed mRNAs
2. Ss-cDNAs from rare mRNAs
3. Partly rehybridized cDNAs from frequently-occurring mRNAs
Selection of the DNA single strands occurred by restriction digestion of the double-stranded cDNAs via "frequent cutter" restriction enzymes. The single-stranded DNAs were reamplified. This normalization procedure was repeated twice.
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