Laboratory approaches in molecular pathology: the polymerase chain reaction

The polymerase chain reaction (PCR) represents a rapid, sensitive, and specific method for in vitro amplification of nucleic acid sequences. Using specific oligodeoxynucleotide primers and a DNA polymerase, the PCR can identify a target sequence and amplify millions of copies (amplicons) of the target. PCR was first described in the mid-1980s. Since then methodological modifications, developments, and new instrumentation have combined to enhance the technology, which has evolved into a reliable, affordable, user-friendly method that is performed in laboratories worldwide. The major technological breakthrough in the development of PCR was the introduction of a thermostable DNA polymerase. The second major technological breakthrough in the development of the PCR was the introduction of the programmable heat block that automatically changes the reaction temperature during each amplification cycle known as the thermocycler. PCR is now used as the basis for numerous clinical molecular diagnostic tests in laboratories worldwide. Real-time PCR, reverse-transcriptase quantitative PCR (RT-qPCR), and digital PCR (dPCR) all allow for the quantification of the target with varying applications that depend on the method's sensitivity. The most recent methodological modification, dPCR provides the absolute quantification and is sensitive enough to detect rare alleles and perform copy number analysis.