cDNA sequencing based on PCR and random shotgun cloning

ID: 84

Group:

Methods for DNA sequencing

Prepared by:

Bruce A. Roe (broe@uoknor.edu)

Detail:

The following is a rapid and efficient method for sequencing cloned cDNAs based on PCR amplification (14), random shotgun cloning (1,3,15), and automated fluorescent sequencing (16). This method was developed in our laboratory because once the sequence of a genomic DNA containing cosmid is obtained and putative exons are predicted, the corresponding cDNAs should be sequenced in a timely manner. However, the presently implemented directed cDNA sequencing strategies, i.e. primer walking (17) and exonuclease III deletion (18), are both time consuming and labor intensive, while the alternative, i.e. randomly shearing the intact plasmid followed by shotgun sequencing (1,3,15), leads to a significant number of clones containing the original cDNA cloning vector rather than the desired cDNA insert.

Year prepared: NULL

Date uploaded: 2010-02-26 14:28:18

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