A novel system for large-scale sequencing of cDNA by PCR amplification

Abstract

We have developed a method for constructing a library containing the 3′ end fragment of cDNA for large-scale sequencing of cDNA clones. The average size of the insert was 270 bp. Cell lysates that carry plasmids having the cDNA insert were subjected to PCR amplification of the cDNA moiety and the products were subjected to sequencing analysis using an autosequencer. With this protocol, sample preparation became a non-limiting step, that allowed us to sequence as many samples as the autosequencer could handle.

Key Words:

• cDNA library
• Hep G2
• large scale sequencing
• PCR artifact
• PCR direct sequencing
• 3′ UTR