Abstract
Publisher Summary The use of degenerate oligonucleotides, that is, a mixture of oligonucleotides of the same length but varying in codon usage, is of great help when searching for new members of a family of genes. However, the use of such oligonucleotides as probes for the screening of cDNA libraries may prove difficult if the clones of interest are present in low copy number, such as the Rab cDNAs. The development of the polymerase chain reaction (PCR) has provided a powerful tool for molecular biology. The use of degenerate oligonucleotides in PCR-based methods can thus be used to identify efficiently new members of a gene family. This chapter details the methods that have used to molecularly clone new members of the Rab subfamily. The strategy involves cDNA amplification by PCR using degenerate primers, followed by the cloning of these fragments into plasmids and their nucleotide sequencing.
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