The polymerase chain reaction (PCR) is a powerful technique that is widely used for amplification of specific DNA sequences in vitro using appropriate primers.1-4 PCR is a major breakthrough technology and is a relatively rapid, sensitive, and inexpensive procedure for amplification and cloning of the cDNA or genomic DNA of interest. It is also invaluable for analysis of RNA expression, genetic diagnosis, detection of mutations and genetic engineering.2-11 The general principles of PCR start from a pair of oligonucleotide primers designed so that forward or sense primer directs the synthesis of DNA towards reverse or antisense primer, and vice versa. During the PCR, Taq DNA polymerase, which is purified from bacterial Thermus aquaticus and is a heat-stable enzyme, catalyzes the synthesis of a new DNA strand complementary to a template DNA from direction by primer extension reaction. This results in production of the DNA region flanked by the two primers. Because the Taq DNA polymerase is high-temperature (95°C) stable, it is possible for target sequences to be amplified for many cycles using excess primers in a commercial thermocycler apparatus. Recently, high-quality Taq polymerases, such as recombinant polymerase Tth, long-spand polymerase and high-fidelity PCR polymerase, have been developed. Due to their capability of proofreading, these polymerases are more advanced enzymes compared with traditional Taq DNA polymerase.

The present chapter focuses on rapid amplification and isolation of specific cDNAs or genomic genes by PCR strategies. Traditionally, cDNA or the gene is isolated from cDNA or genomic DNA libraries, which involves construction and screening of cDNA or genomic DNA libraries. The procedures are complicated, time-consuming, and costly. Besides, it may be impossible to "fish" out the low copy cDNAs transcribed from rare mRNAs in cDNA libraries. In contrast, the rare cDNA can be rapidly amplified and isolated by the reverse transcription polymerase chain reaction (RT-PCR). This chapter describes detailed protocols for fast isolation and purification of the cDNAs or genes of interest. These protocols have been successfully used in our laboratories.

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