Digital cloning: identification of human cDNAs homologous to novel kinases through expressed sequence tag database searching.
Identification of novel kinases based on their sequence conservation within kinase catalytic domain has relied so far on two major approaches, low-stringency hybridization of cDNA libraries, and PCR method using degenerate primers. Both of these approaches at times are technically difficult and time-consuming. We have developed a procedure that can significantly reduce the time and effort involved in searching for novel kinases and increase the sensitivity of the analysis. This procedure exploits the computer analysis of a vast resource of human cDNA sequences represented in the expressed sequence tag (EST) database. Seventeen novel human cDNA clones showing significant homology to serine/threonine kinases, including STE-20, CDK- and YAK-related family kinases, were identified by searching EST database. Further sequence analysis of these novel kinases obtained either directly from EST clones or from PCR-RACE products confirmed their identity as protein kinases. Given the rapid accumulation of the EST database and the advent of powerful computer analysis software, this approach provides a fast, sensitive, and economical way to identify novel kinases as well as other genes from EST database.
Chen
H C
HC
Molecular and Genomic Medicine Division, National Health Research Institutes, Taipei, Taiwan, ROC.
Kung
H J
HJ
Robinson
D
D
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D016208:Databases, Factual; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011494:Protein Kinases