( frequently occurring cDNAs...)Normalizing cDNA libraries using the EppendorfThermomixer

frequently occurring cDNAs, and an increase in rare cDNAs. This facilitates access to the group of "least abundant" mRNAs. The difference in the number of clones, which often varied by a factor of 20,000, was drastically reduced by this normalization procedure to a factor of 40.

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:

Classical Method:

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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