If different primers are to be used for the upstream and downstream positions, synthesis of cDNA should be driven with the downstrean primer. A strategy has been devised to generate different populations of cDNAs by using "tailed" oligodT primers for both the synthesis of cDNA and the PCR process. This method, developed by Liang and Pardee (4), relies on the use of several downstream primers in combination with multiple upstream primers. This approach allows a myriad of combinations. Furthermore, additional products can form as a result of the priming of the individual primers with themselves. The cost of synthesizing and purifying the multiple primers used in this method can be considerable, and the authors have now commercialized a kit (GenHunter Corporation, 50 Boylston St., Brookline, MA 02146) that is definitively worth trying. This method has been proven successful in a variety of systems and could be a good choice. The major disadvantage is that this method will select preferentially for abundant mRNAs, and that the products will belong preferentially to the 3' noncoding regions that are the least conserved among related genes in different species. This can make data base searching less informative.
Both methods have been proven succesful in a variety of biological systems (4,6-9). Unfortunately, it is impossible to predict what will be the result of choosing one strategy over the others until the experiments are actually done and the products analyzed. Once an authentic product has been isolated, isolation of a full-length cDNA will require the screening of a suitable cDNA library, although other PCR-based methods can also be used for this purpose (10).
Here, we provide a protocol that can be followed among many others. This protocol is based on the RAP-PCR approach, as described originally by Welsh et al. (3). A very detailed protocol of the second method has been described (4), or can be obtained from GenHunter Corporation. Overall, what is really needed is not many protocols but a great deal of good luck.
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