Is nested PCR necessary for the RACE strategies?
The RACE strategies that are used to identify unknown sequences at the terminal ends of the cDNA inserts are technically challenging. Since these strategies utilize a vector sequence primer common to all cDNAs in the library, it is critical to insure that the internal primer is gene-specific and well designed to avoid mispriming of unintended sequences. For this reason, it is essential to employ a nested strategy for the internal primers and to utilize the BLAST (Basic Local Alignment Search Tool) tool at http://blast.ncbi.nlm.nih.gov/Blast.cgi to insure that the primer sequences do not hybridize with other gene sequences. This will help considerably in the PCR optimization and in obtaining clear results without PCR artifacts. Because of the technical challenges with RACE, we don’t recommend this strategy to be used on the entire 96-well Splicist panel. However, it can be implemented successfully to further investigate sub-pools previously found to be positive for your gene of interest
