Amplicon Carryover Contamination

Vast numbers of target DNA copies are generated when PCR and other in vitro nucleic acid amplification techniques are used. By contrast, signal amplification methods (see below) do not generate vast quantities of amplicon and so do not create the potential for amplicon carryover contamination of the laboratory workspace. Amplicons from previous reactions inadvertently introduced into new amplification reactions for the same amplicon are suitable substrates for amplification. Clinical molecular laboratories must therefore take precautions to prevent generation of false-positive results from amplicon carryover contamination.

Amplicon contamination and false-positive results are prevented by using physical barriers and chemical and ultraviolet techniques to destroy amplicons or make them unsuitable for amplification. The physical barriers include large-scale separation of nucleic acid isolation, PCR setup, thermal cycling, and post-PCR analysis in separate areas of the laboratory (different rooms). Air flow is controlled such that air pressure is positive, that is, flows out of the room, in the isolation and PCR setup rooms and is negative in the thermal cycling and post-PCR analysis rooms. Hoods are another way of providing physical separation of the differ ent PCR steps. Small-scale physical separation techniques include the use of barrier pipette tips, frequent glove changes, designated lab coats that do not leave the pre- or post-PCR areas of the laboratory, and PCR tube openers or careful, slow opening of tubes to prevent aerosolization of contents. Real-time PCR reduces the chances of amplicon contamination since the PCR product can be detected and quantified without opening the real-time PCR reaction vessel after PCR.

Chemical techniques include thorough cleansing with bleach of work areas and instruments before and after use. Ultraviolet lights are frequently placed in hoods and work areas. Ultraviolet light creates thymine dimers within amplicons, rendering the amplicons unsuitable as substrates for further amplification. The introduction of isop-soralens in PCR reactions allows DNA cross-linking of amplicons by UV light, also rendering them unsuitable for further amplification. Deoxyuridine may be used in lieu of thymidine in the reaction mixture. Use of deoxyuridine has minimal effect on amplification or product detection, but amplicons with uracil are substrates for uracil-N-glycosy-lase (UNG). UNG has no effect on DNA that contains only thymidine residues (new patient DNA in subsequent reactions) but digests the uracil-containing amplicons, allowing removal of amplicons before PCR proceeds.62 So-called UNG sterilization may therefore be performed prior to PCR to rid the reaction of any amplicon contaminants that may be present.

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