A rapid protocol for the authentication of isolated differential display RT-PCR CDNAs

摘要

The most time-consuming and problematic step in the overall DDRT-PCR technique is the confirmation that the isolated cDNA clone represents a differentially expressed gene. We have previously suggested that the majority of apparent false positives generated by DDRT-PCR do in fact result from the PCR reamplification of cDNA species which co-migrate with the cDNA of interest, and we have outlined a procedure to effectively eliminate these from further study. However, in situations where RNA is limiting, it is still desirable to confirm that a purified cDNA amplicon does, in fact, represent the originally observed differentially expressed gene prior to embarking on expression studies. The protocol presented here allows rapid verification of isolated Differential Display RT-PCR cDNA clones. We outline how a simple Southern blotting procedure can be used prior to labor-intensive expression studies to validate that a particular isolated cDNA clone does represent the original band of interest. We also describe the use of modified oligonulcleotides for PCR reamplification of isolated DDRT-PCR cDNAs which allows efficient and unbiased cloning into inexpensive, widely available cloning vectors.