Setting up a PCR laboratory. (1993)

PCR technology now permeates all of molecular biology.For many PCR applications, it is essential that the only DNA that enters the reaction is the template added by the investigator.The ideal way to ensure that PCR is performed using only the desired template is to maintain a DNA-free, clean environment.The issue of contamination and the cleanliness required to perform contamination-free PCR has been compared to the good microbiological techniques essential for handling pathogens.~ The major difference for PCR is that the "biohazard" infects your PCR, not the researcher.In this section we will describe some general guidelines for the establishment and maintenance of a clean environment for any PCR-based assay system, regardless of the number of samples being processed.As the use of PCR grows in areas such as clinical diagnosis of genetic diseases (2) or the monitoring of viral burden in patients on antiretroviral therapy, (3) rational guidelines for installing a PCR facility will be essential.Our discussion will deal with the establishment of two types of laboratories, one performing contamination-sensitive PCR assays, such as measurement of quantities of target sequences in a sample, and those that are using PCR as a contaminationinsensitive molecular biology tool, such as for mutagenesis of a DNA clone.The purpose of considering the design, location, and execution of PCR in discrete areas is to plan ahead to avoid contamination of the new PCR reactions with either old PCR products, molecular clones, or sample-to-sample contamination.To date, three approaches have been devised to prevent contamination.The first is the physical separation of the individual parts of the PCR reaction into sample preparation, a pre-PCR location, and a post-PCR location.(4) This approach requires space, money, and supplies to equip and maintain a large infrastructure.However, this is not foolproof because good laboratory practice is still required for the prevention of sample-to-sample contamination.The second method, the use of uracil DNA-glycosylase (UDG) and dUTP substituted for dTTP, is effective only against contamination with dUTP-labeled PCR products.(s) The final method, the use of UV light, is effective against all types of contamination.Its limitation is that this approach cannot destroy all of the contamination, and only reduces it by several logs.Also, it is less effective if the DNA fragment is < 300 bp.(6'7) With these caveats in mind, we suggest the following guidelines for establishing a PCR laboratory. ESTABLISHMENT OF A PCR LABORATORYTo perform PCR for the repetitive detection of a specific sequence, three distinct laboratory areas are required.The specific technical operations, reagents, and personnel considerations for each one are detailed below.There is new interest in using PCR for the quantitative detection of target sequences, primarily HIV.(3) Thus, as the importance of viral burden measured by RNA-PCR grows, the need for contamination-free, reliable PCR becomes essential. Sample Preparation AreaThis room is specific for sample preparation only.No handling of PCR products or DNA clones containing the sequence to be amplified can take place within this room.Tissue cultures, tissue specimens, and serum samples are all brought into the room and processed for the extraction of DNA or RNA, depending on the application.The tools used in sample processing should not be used for general molecular cloning or manipulation of the target sequence.Aerosols are minimized by briefly centrifuging the tubes prior to opening.Also, the tubes should not be popped open, as this can create an aerosol.The DNA samples should be manipulated with specialized barrier or positive-displacement pipettes, which will prevent the carryover of aerosols $2 PCR Methods and Applications 3:S2-S7 9 by Cold Spring Harbor Laboratory