Rapid isolation of desired sequences from lone linker PCR amplified cDNA mixtures: application to identification and recovery of expressed sequences in cloned genomic DNA.
A simple and efficient method for the rapid isolation of specific sequences from PCR-amplified cDNA mixtures has been developed. cDNA mixtures obtained using lone linker PCR (Ko et al. 1990) appeared to be highly representative even though the starting material, 100 ng-2 micrograms of total RNA, is much less than is required for making an ordinary cDNA library. With this method, cDNA mixtures were obtained from limited materials, including early mouse embryos and primordial germ cells. For selective enrichment of desired cDNAs, biotinylated probe was hybridized with the lone linker-linked cDNA in solution and the resulting probe-cDNA hybrid was captured by Streptavidin-coated magnetic beads. After appropriate washing, cDNA was released from the beads and subjected to amplification followed by cloning into a vector. Using genomic fragments isolated during chromosomal walking in the T/t complex of mouse Chromosome (Chr) 17, cDNAs encoding novel germ cell specific genes have been readily isolated by the above procedures. The method, termed random access retrieval of genetic information through PCR (RARGIP), will streamline the entire process from RNA to cDNA greatly. Its application potentials in various areas of molecular genetics will be discussed.