E-Book Overview
Ray H. Gavin brings together an international panel of experienced researchers to detail the readily reproducible methods that utilize biochemistry, immunology, genetics, microscopy, and image analysis for investigating cytoskeleton structure and function. Each protocol contains proven step-by-step instructions that enable both the novice and the experienced researcher to achieve successful experimental results. The protocols utilize diverse model systems in a variety of organisms, including Saccharomyces, Micrasterias, Tetrahymena, Drosophila, Spisula, and Xenopus. Microscopy applications include digital-video microscopy and computer-assisted systems for the evaluation of cell motility and morphology. State-of-the-art and highly practical, Cytoskeleton Methods and Protocols makes available a diverse collection of powerful experimental systems and tools for successfully studying cytoskeleton structure and function.
E-Book Content
Methods in Molecular Biology
TM
VOLUME 161
Cytoskeleton Methods and Protocols Edited by
Ray H. Gavin
HUMANA PRESS
Inverse PCR
3
1 Using an Inverse PCR Strategy to Clone Large, Contiguous Genomic DNA Fragments Jorge A. Garcés and Ray H. Gavin 1. Introduction 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. However, 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 conventi