Extension of incomplete cDNAs (ESTs) by biotin/streptavidin-mediated walking using the polymerase chain reaction

Abstract

In the last 2 years thousands of new partial cDNAs or expressed sequence tags (ESTs) have been identified by single pass sequencing methods. It is expected that this number will further increase in order to help to isolate all human genes. However, the scientific value of partial cDNA fragments is limited unless they are used as tools for isolating and sequencing their full length parent molecules. Conventional library screening methods are tedious and not very effective in achieving this goal. We present a modified PCR technique which allows rapid isolation of the ends of partial cDNA fragments in vitro using a biotin/streptavidin capture procedure. Our method has several advantages over the RACE technique, is very specific, and allows to frequently sequence the final product directly without subcloning. We also show that cDNA walks can be obtained from partial sequences as short as 26 bp.