Authors
Creative Bioarray
Summary
Polymerase chain reaction (PCR) represents a rapid, sensitive, and specific method for in vitro amplification of nucleic acid sequences. By using specific oligodeoxynucleotide primers and DNA polymerases, the PCR is able to identify a target sequence and then amplify millions of copies of the target.
Introduction
As a molecular technology, PCR facilitates the amplification of specific nucleic acid sequences to produce a large number of amplified products that can be analyzed by other methods. Therefore, the PCR offers a very sensitive and specific method for the quantitative and qualitative analysis of target sequences. The development of various chemistries for primer and probe labeling has produced an extraordinary technology in terms of performance characteristics.
Procedure
Hot-start PCR
Hot-start PCR was developed to reduce background from nonspecific amplification. Hot-start prevents new DNA from polymerizing during the initial phase of the reaction when nonspecific binding may occur between primers and nonspecific DNA targets.
Nested PCR
This is a form of re-amplification of the PCR product. Nested PCR increases the specificity of the reaction since formation of the final product relies on the binding of two separate sets of primers and two sets of amplification.
Reverse Transcriptase PCR (RT-PCR)
This begins with the conversion of RNA to complementary DNA (cDNA) by a reverse transcriptase. RT-PCR is an excellent method for analyzing RNA transcripts, especially for measuring low-abundance species or working with limited amounts of starting material. Since RNA is not as stable as DNA, fresh samples are generally required for RNA analysis.
Real-time PCR (Q-PCR)
Real-time PCR (Q-PCR) allows for the real-time quantitation of PCR product following each amplification cycle. The technique of Q-PCR therefore offers a great rapid quantitative advantage. Moreover, it is less prone to contamination since the entire process of amplification and quantitation of the original target DNA for each sample is performed in a single sealed tube.
Multiplex PCR
This involves the addition of several different primer pairs to the same reaction mixture to detect multiple abnormalities or infectious agents in the same assay. Multiplex PCR is particularly useful for detecting mutations, deletions, insertions and rearrangements in clinical specimens.
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