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polymerase chain reaction (PCR)
Procedure:
- Amplification of DNA through action of cycles of denaturation of DNA, annealing of primers to DNA, & DNA synthesis using thermostable DNA polymerases.
- Theoretically, for n cycles of replication, the DNA may be amplified 2(exp)n fold.
- Careful selection of primers complementary to unique DNA sequences on the amplified DNA is necessary.
Specimen:
1) 10 ng of DNA is adequate for quantitative PCR measurements
2) crude cell lysate, serum & whole blood may be used directly
3) avoid anticoagulants
a) heparin is a strong inhibitor of PCR
b) citrate & EDTA are acceptable anticoagulants
4) heme is a strong inhibitor of PCR
5) specimens should be stored at -20 degrees C or below
Interferences:
- common, including
a) carry-over exogenous DNA from contamination of glassware
b) DNA on skin
c) aerosolized DNA
Negative controls are of critical importance.
Notes:
- Turnaround time: 4-6 hours
Related
molecular diagnostic test
Specific
multiplex polymerase chain reaction (PCR) assay
reverse transcriptase polymerase chain reaction (RT-PCR)
telomeric repeat amplification protocol (TRAP)
General
special chemistry test
References
- Medical Knowledge Self Assessment Program (MKSAP) 11, American
College of Physicians, Philadelphia 1998
- Clinical Guide to Laboratory Tests, 3rd ed. Teitz ed.,
W.B. Saunders, 1995