Polymerase Chain Reaction

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Contents 1 More Specific Terms 2 Specimen 3 Procedure 4 Interferences 5 Notes 6 More General Terms 7 Additional Terms 8 References

More Specific Terms

• reverse transcriptase polymerase chain reaction (RT-PCR)
• telomeric repeat amplification protocol (TRAP)

Specimen

• 10 ng of DNA is adequate for quantitative PCR measurements [3]
• crude cell lysate, serum & whole blood may be used directly
• avoid anticoagulants

• heparin is a strong inhibitor of PCR
• citrate & EDTA are acceptable anticoagulants

• heme is a strong inhibitor of PCR
• specimens should be stored at -20 degrees C or below

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.

Interferences

• common, including

• carry-over exogenous DNA from contamination of glassware
• DNA on skin
• aerosolized DNA

• Negative controls are of critical importance.

Notes

• Turnaround time: 4-6 hours

More General Terms

• special chemistry test

Additional Terms

• molecular diagnostic test

References

1. Medical Knowledge Self Assessment Program (MKSAP) 11, American College of Physicians, Philadelphia 1998
2. Clinical Guide to Laboratory Tests, 3rd ed. Teitz ed., W.B. Saunders, 1995
3. Dan Geschwind, UCLA Dept of Neurology, personal communication

polymerase chain reaction (PCR)