Discriminating PCR Artifacts Using Directed Heteroduplex Analysis (DHDA)

摘要

The polymerase chain reaction (PCR) has been applied to an ever-expanding number of biological studies. However, this powerful tool is often subject to errors. Two types of PCR artifacts are commonly found. The first (Type 1) involves amplification ofunwanted sequences by the primers. Although this type of artifact does not occur on a regular basis and is largely dependent on the primers and reaction conditions used, it can be a nuisance when a large number of PCR products need to be analyzed. The second type of artifact (Type 2) results from the mis-incorporation of nucleotides during the amplification process and may be attributed to infidelity of the thermostable DNA polymerases in vitro. Type 2 artifacts are most troublesome when cloned examples of the PCR product are required for other studies, such as overexpression of open reading frames contained in the PCR product. To ensure that PCR mutations have not been introduced into cloned products, it is necessary to determine the sequence of multiple cloned PCR products, a process that is both time-consuming and expensive.