Cytoskeleton Methods and Protocols

Conventional PCR screens of genomic DNA will often yield a substantial fragment of the gene of interest. However, identification of flanking sequences on either side of a known sequence can be problematic with conventional PCR, in which primers extend a complementary chain in a 5' → 3' direction. How- ever, if the template DNA is circularized and then used with primers that are oriented with their 3' ends directed away from each other, amplification around the circular template results in a linear PCR product consisting of uncharacterized DNA fragments flanked by the known DNA sequences (Fig. 1). This variation of the conventional PCR strategy is known as inverse PCR (1,2), and we have used the technique to amplify myosin sequences in Tetrahymena (3). In this chapter we describe protocols that enable the investigator to apply the inverse PCR technique to clone contiguous sequences upstream and down- stream of a known DNA sequence.