Rapid isolation of long cDNA clones from existing libraries.

Obtaining full-length or even near full-length cDNA clones has been a time-consuming and labor-intensive step in the analysis of cloned genes. Recent methods which use PCR as a preparative tool for cloning (1,2) have made this step considerably more rapid, but may introduce sequence errors in the resulting clones due to numerous sequential rounds of in vitro replication. We describe a method for identifying long cDNA clones from existing cDNA libraries using PCR purely as a diagnostic tool. The method is shown schematically in Figure 1. Sequence information from the 5' end of an existing clone is used to design a gene-specific PCR primer. This is used in combination with a primer that abuts the 5' cloning site in the vector to amplify a directionally cloned cDNA insert and then to reamplify the insert in the presence of ^P and the absence of significant vector sequences to generate a radiolabeled 5' end probe for screening libraries by plaque filter hybridization (3). For efficiency, we