Combination of Direct DNA Sequencing with Degenerate Primer-Mediated PCR and 5'-/3'-RACE to Screen Novel cDNA Sequences

摘要

Cloning of new DNA sequences using amino acid sequence data is a cumbersome and time-consuming procedure that usually involves screening of the cDNA or genomic libraries with ~(32)P-labeled probes or antibodies, sub-cloning the positive clones, purification of plasmid DNA and, finally, sequencing of inserted DNA fragments. Poly-merase chain reaction (PCR)-mediated amplification of cDNA with degenerate oligonucleotide primers allows isolation of the corresponding cDNA without screening libraries by traditional hybridization.