) The methodologies for the preparation of cDNAs and for the cloning of these molecules into vectors has advanced over the last ten to fifteen years, however, the production of long cDNAs remains problematic. One of the major impediments to synthesizing long cDNAs is thought to be the presence of secondary structure in the mRNA that prevents or inhibits the progression of reverse transcriptase enzymes. The advent of reverse transcriptases that function at temperatures much higher than conventional enzymes has led to the prospect that the enzymes could function under conditions that would alleviate mRNA secondary structure. Unfortunately, these enzymes have proven to be of a not very highly processive nature. We have, therefore, developed a procedure that relies on both a conventional enzyme for synthesizing long stretches of cDNA and a high temperature enzyme to bypass secondary structure by incubation at high temperature. By cycling between temperatures for a number of times we show preliminary evidence that we can prepare long cDNAs in a simple and robust procedure. In this application we are proposing to further develop this approach and use it for the preparation of cDNA libraries for different stages of colon cancer tumors and corresponding normal tissue.
