Encyclopedia of Experiments: Biological Techniques
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Transcript
Nested polymerase chain reaction, nested PCR, can selectively amplify specific gene sequences.
To detect a target viral sequence, begin with a master mix containing outer primers complementary to a larger DNA region containing the targeted viral sequence, dNTPs, and thermostable DNA polymerase in an appropriate buffer.
Add double-stranded viral DNA, extracted from the virus-infected cells, to this mix. Transfer the tube to a thermocycler; run the first PCR cycle.
During denaturation, the double-stranded DNA produces two single-stranded templates. At an appropriate annealing temperature, outer primers bind to their corresponding target sequences. Later, DNA polymerase extends the primers using dNTPs, producing large amplicons, containing the specific sequence within the amplified DNA.
Transfer these amplicons to a new tube. Supplement with a fresh master mix containing inner or nested primers targeting a smaller, more specific sequence within the larger amplified DNA. Run the second PCR.
During the second PCR, the DNA strand denatures. After denaturation, the nested primers bind the target sites on the single-stranded template, followed by polymerase-mediated primer extension.
Repeat the second PCR cycle several times to exclusively amplify the targeted viral sequence.
Analyze the PCR product to visualize the smaller amplicons, confirming the presence of the specific viral sequence.
