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Indiana University Bloomington

PCR/RT-PCR

The polymerase chain reaction (PCR) is a common molecular technique used to exponentially amplify a DNA sequence of interest. Amplification is possible even with tiny amounts of starting material. When it is cDNA that is amplified (the “complement” to a mRNA sequence), it is termed reverse-transcription or RT-PCR. PCR technology is used for a variety of applications, including DNA sequencing, genotyping (for genetic profiling or phylogenetics) and gene expression analysis.

PCR relies on the use of primers, which are short, single-stranded nucleotide sequences (oligonucleotides) used to “target” the beginning and end of your DNA sequence of interest (product). In brief, the PCR process involves: (1) denaturing the sample to yield single-stranded DNA; (2) annealing of primers to the single-stranded DNA at the sequence of interest; and (3) extension/elongation of the DNA sequence of interest to create two double-stranded DNA copies. This process is then repeated several times to exponentially increase the number of copies of your desired product.

More information about the theory and practice behind PCR and RT-PCR can be found at the NIH website by entering “pcr” into the search option.

PCR and RT-PCR protocols often require optimization to determine the correct temperatures, timing and number of cycles for denaturing, annealing and extending your product. These protocols also require validation when a primer set is used for the first time, to make sure the primers are recognizing the sequence you want. Designing primers that accurately isolate your DNA or mRNA sequence of interest can be tedious, but there are many computer programs available to aid in this process.

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