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Updated: Jul 5, 2026

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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Published on: May 22, 2012
The polymerase chain reaction
Martha F Kramer1, Donald M Coen1
1Harvard Medical School, Boston, Massachusetts.
Current Protocols in Protein Science
|April 23, 2008
Abstract:
This appendix describes the methodology behind PCR and gives a method for amplifying DNA enzymatically by the PCR and for optimizing this reaction for the sequence and primer set of interest.
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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...
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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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