With the genomic sequence of multicellular organisms such as Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens completed and others to be finished in the near future, the focus has shifted from accumulating sequence information to the prediction and analysis of genes within the completed genomes. Unfortunately, presently available computer programs do not always accurately predict gene structure such as mRNA and translation start sites or intron/exon boundaries. The only way to be certain about a gene's structure is to isolate and characterize its cDNA. Since the screening of libraries is a time-consuming, labor-intensive process that sometimes fails to yield the desired clone, we searched for faster, more efficient ways to isolate cDNAs. In this study, we describe two methods for amplification and isolation of cDNAs from plasmid libraries that requires no hybridization (MACH). With the polymerase chain reaction-based MACH-2 protocol, we present a strategy that requires little DNA sequence information to selectively isolate the longest cDNA variant from plasmid libraries in about 3 days. Our protocols were used to isolate cDNAs for the Drosophila activin and follistatin genes.
Bibliographical noteFunding Information:
We would like to thank Guillermo Marqués for reading the manuscript and members of the O'Connor lab for additional testing of these protocols. This study was supported by grant PHS (GM47462) to M.B.O. Michael B. O'Connor is currently an Associate Investigator with the Howard Hughes Medical Institute.
- Polymerase chain reaction
- cDNA cloning