Verification Of Pcr Products

Although DNA bands of the expected size on an agarose or polyacrylamide gel are an encouraging sign that the reaction produced the desired amplicons, the identity of the PCR product should be verified by a secondary method. Verification is usually accomplished by nucleotide sequencing, restriction mapping, or sequence-specific probe hybridization (Fig. 3). Sequencing a PCR product provides the best information on the identity of the amplicon and represents the best method for verification of PCR products. Typically, the PCR product is cloned and sequenced by standard methods for either double-stranded or single-stranded DNA. With the advent of microsequencing techniques, purified products can be sequenced without having to be cloned (32,33), shortening the time needed to verify a product considerably by eliminating the cloning step. Restriction mapping of PCR products is probably the simplest verification method. To use this method, the amplified sequence must contain one or more known unique restriction sites. Thus, products can be verified by digestion with the appropriate restriction endonuclease and subsequent separation and visualization by gel electrophoresis. Sequence-specific hybridization can also be used to verify PCR products. In this method, a synthetic oligonucleotide that recognizes a unique sequence within the amplified region is hybridized to the PCR product. Successful hybridization is measured by autoradiography (or related technique) and the resulting autoradiogram can be used to quantitate the amount of product produced. This method has the added advantage that stringent hybridization and washing conditions can be used along with other probes to differentiate between related gene transcripts that are similar in size.

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