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[28] Sequencing of polymerase chain reaction-amplified DNAs

1993; Academic Press; Linguagem: Inglês

10.1016/0076-6879(93)24029-t

1557-7988

W. Kelley Thomas, Thomas D. Kocher,

Identification and Quantification in Food

This chapter describes various methods for generating templates suitable for sequencing from polymerase chain reaction (PCR) products. Three general approaches are discussed: (1) the cloning of PCR products, (2) preparation of single-stranded templates from PCR products, and (3) direct use of double-stranded PCR products as sequencing templates. The enzymatic amplification of a specific segment of DNA (target sequence) by the PCR has several advantages over traditional, in vivo methods of cloning DNA. The PCR is much more rapid than traditional library construction and screening because it involves far fewer manipulations. In addition, smaller regions of sequence similarity can be used to retrieve a particular sequence and it is possible to amplify from relatively crude preparations of DNA and from degraded DNA samples. The sensitivity of PCR allows amplification of as little as a single molecule of DNA, which has permitted the recovery of sequences from unculturable bacteria and even single sperm. Finally, in many cases, direct sequencing of PCR products offers the opportunity to determine DNA sequences with greater accuracy than traditional cloning methods.