Molecular - PCR - Nested PCR

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Molecular Pathology

Polymerase Chain Reaction

Nested PCR

Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)

Revised: 8 July 2010, last major update July 2010

General

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● Nested PCR uses two sets of primers, one set is internal to, or “nested within” with other set

● The first or “outer” pair of primers amplifies the target sequence; then a dilution is made of the reaction, and the internal or nested primers are used to continue the amplification

● Alternatively, the technique can be “semi-nested” where one original PCR primer and one internal primer are used after the first amplification

● Both techniques will generate an amplified sequence shorter than the first amplified sequence

● If the first reaction is amplified nonspecifically, then it is unlikely that the internal amplification would also proceed

● Thus, this technique increases the specificity and sensitivity of the PCR reaction

● A drawback of this technique is that the addition of new primers after the first amplification round increases the chances of nonspecific contamination; many clinical labs avoid this technique for this reason

End of Molecular Pathology > Polymerase Chain Reaction > Nested PCR

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