Current PCR Methods

 Introduction

History

The Polymerase chain reaction (PCR) is a ubiquitous technique utilized extensively for diagnostic purposes and molecular biology research. PCR is the in vitro amplification of specific nucleic acid (NA) sequences by a DNA Polymerase enzyme. The PCR technique was transformed by Kary Mullis in 1983, when he expanded the use of a heat-stable Polymerase with temperature cycling [1] [2] [3] . The universal utility of PCR is that it amplifies small quantities of target nucleic acid sequences, yielding an amount of product that is detectable by downstream methods, such as visualization of NA on an agarose gel. This is due to the exponential amplification of the sequence and the resulting millions of copies of the original template.

The Basic PCR Reaction

PCR reactions amplify target nucleic acid sequences via the use of a DNA Polymerase, primers, and nucleotides. The template for a PCR reaction may be any nucleic acid sequence of interest, and the NA source may be DNA, RNA, or cDNA. Primers are short sequences of nucleotides synthesized in vitro. They are designed to anneal to opposite strands of a specific NA template target, and are normally between 15-40 bases long. Primers ideally lack secondary structure and are not complementary to each other, to prevent primer dimer formation. A variety of DNA Polymerases have been utilized for PCR, but the thermostable Taq DNA Polymerase is probably the most widely used. This enzyme adds the deoxyribonucleoside triphosphates (dNTPs or nucleotides) onto the ends of the primers to extend the NA based on the template NA sequence.

The PCR reaction mixture is temperature cycled, normally 20-40 times. Denaturation of the NA template sequence is achieved at 95ºC. To anneal primers to the target sequence, the temperature is cooled to 37-60ºC. Extension of the primers with nucleotides by the DNA Polymerase is achieved at temperatures ranging from 60-72ºC. Conventional cycling conditions are 95ºC for 5 minutes initially to denature all template NA, followed by 2-40 repeats of 95ºC for 30 sec, 60ºC for 30s and 72ºC for 1 minute. The time spent at each temperature can be optimized for specific assays. For instance, the amplification of very short target sequences requires much shorter incubations at each temperature than that of very large target sequences. Each round of temperature cycling results in two times more target sequence than the prior round. This leads to the exponential amplification of the original template, often resulting in millions or billions of copies of the original NA target.

The basic PCR reaction occurs in three phases. The exponential phase is the period in which exact doubling of nucleic acid product occurs every cycle. Real-time PCR detection is carried out during this exponential phase. The linear phase occurs as the reaction is slowing due to the consumption of the reagents and the degradation of the products. The final stage is the plateau phase, which occurs when the reaction has stopped and no additional amplicon is being generated. This is the point at which the PCR reaction product is analyzed via gel electrophoresis for conventional PCR reactions.

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