QUALITY ASSURANCE IN PCR LABORATORY BY CONTAMINATION CONTROL

QUALITY ASSURANCE IN PCR LABORATORY BY CONTAMINATION CONTROL

Development of the polymerase chain reaction (PCR) as a basic component of the molecular biology laboratory has occurred very rapidly from its inception in 1985. Since then, more than 15,500 articles have been published in which this technique was used. As PCR became more widely used, scientists rapidly learned more about it and, as a result, learned that the PCR had its strong points and its deficiencies. Very quickly, PCR demonstrated its power to amplify very small amounts (e.g., a single copy) of template nucleic acid and to amplify different nucleic acids (e.g., DNA and RNA). At the same time, laboratory personnel learned that this biochemical reaction had a unique deficiency; namely, a strong susceptibility to contamination from its own product. PCR contamination remains an issue for laboratories performing forensic procedures and detection of infectious agents (Pellett et al. 1999; Scherczinger et al. 1999). There are a number of approaches to control of PCR contamination, and the degree of stringency that is required in a laboratory is often determined by the assay being performed.

Sources of Contamination:

Amplicon Aerosol

The single most important source of PCR product contamination is the generation of aerosols of PCR amplicons that is associated with the post-PCR analysis. Methods for eliminating this aerosol range from physical design of laboratories and use of specific pipettes to chemical and enzymatic approaches.

Target Template Contaminants

In addition to post-PCR contamination, the target template itself can be a source of contamination. For example, DNA templates are typically more troublesome as contaminants because they are more stable than RNA targets.

Real-time PCR Systems

PCR systems exist that provide direct measurement of amplicon accumulation during the reaction. These real-time PCR systems offer an alternative approach to the traditional post-PCR analysis methods. From a contamination control perspective, the collection of data during the amplification reaction by using a fluorescence-based detection system eliminates the need to handle the sample. Thus, when these PCRs are completed, the detection and analysis are complete, the reaction tubes remain sealed, and there is no amplicon escape

Previous Amplification And Purification Of Plasmid Clones

Repeated Isolation Of Template (Genomic) Nucleic Acids

A Previously Amplified Molecules (“Amplicons”)

Specimen Carryover: The Major Pitfall of PCR:

Careful attention to technique is essential to performing PCR. Because of the high sensitivity of the procedure, the introduction of even a minute amount of a positive template into a negative specimen can lead to the generation of a false-positive result

Designing of PCR Laboratory:

The PCR laboratory typically is involved with activities that include sample preparation, PCR reaction assembly, PCR execution, and post-PCR analysis. When arranged in this linear fashion, these activities can be collected into two major groups, the pre-PCR activities (sample preparation and PCR preparation) and the post-PCR activities (PCR execution and analysis). The essential parts of this contamination control program include space and time separation of pre- and post-PCR activities, use of physical aids, use of ultraviolet (UV) light, use of aliquoted PCR reagents, incorporation of numerous positive and negative or blank

PCRs (H2O substituted for template), and use of one or more various contamination control methods that use chemical and biochemical reactions. The underlying theme in these actions is the recognition that amplicon contamination cannot be seen, felt, or a priori detected before it happens. Use of consistent, careful technique coupled with liberal incorporation and monitoring of PCR blanks will ensure a vigilant, proactive approach to PCR contamination.

The PCR laboratory should consist of three distinct work areas. In order to avoid the contamination problems, each area should be dedicated to a single procedure. Specimen preparation occurs in the first area, reagent preparation and PCR set-up in the second area, and amplification and detection in the third area.

The entire procedure can be performed in a single room if proper precautions are taken. The following practices will diminish the potential for contamination.

v  Each area should have dedicated supplies and reagents.

v  Color coding of reagents and supplies identifies those that belong to a particular area.

v  Reagents, supplies and equipment should never be taken from one area to another three sets of pipettors are therefore.

v  The worlflow must be unidirectional from "clean" (pre-PCR) to "dirty" (post-PCR).

v  Dedicated labcoats and gloves should be worn at each worksite; when moving to a new area, workers should put on new gloves and labcoats.

Space and Time Separation

As illustrated in Figure 1, the main source of the feedback contamination is the amplicons generated by the previous PCR. By separating the source of the amplicons’ (e.g., post-PCR) activities from the pre-PCR activities, the potential for contamination is significantly reduced. This separation is best illustrated by separating the facilities in space, so that there are two rooms where these activities occur. If this is not achievable, different areas designated for sample preparation and PCR setup can be located away from the area for post-PCR analysis. If all activities are to be performed in a single room, sample preparation should occur inside a laminar flow hood, preferably equipped with a UV light. The walls of the hood should be wiped with a fresh (or freshly made) 10% bleach solution (1 part regular bleach: 9 parts water) before processing samples or preparing PCR samples. Waste materials that contain PCR amplicons should not be allowed to accumulate in an area that is also frequented by other personnel who may be eventually involved with template isolation and purification. Additionally, the laboratory should consider establishing a daily schedule for performing PCR. Sample preparation and pre-PCR should be morning activities. Once completed, the pre-PCR supplies and equipment can be stored, and the afternoon can be devoted to the post-PCR analysis.

Laboratory Space Arrangement

As mentioned above, the ideal arrangement of the PCR facility is to have the pre- and post-PCR areas located in separate rooms, each with dedicated resources. A source of deionized water needs to be present in both rooms, as well as dedicated centrifuges, storage freezers /refrigerators, and storage of supplies. Even telephones, computers, and other electronic communications should also be dedicated.

Equipment in PCR Laboratories

To ensure that pre-PCR and post-PCR events remain separated, each room must have its own separate set of equipment, including pipettors, reagents, pipettor tips, racks, and so forth. Moreover, these items should not leave the area to which they are assigned. Each should be labeled as to location and used in that location only. Lab coats should be dedicated for both areas as well. Because pipetting forms the basis for most PCR analysis, each area needs its own dedicated pipettors that are never exchanged between work areas. To assist with this, color-coded pipettors (e.g., green for pre-PCR work, red for post-PCR work) can be used. When pre-PCR pipettors and tips are not in use, they should be stored in airtight bags to keep them clean. Reactions should be constructed using master mixes, and the template should always be added last using positive displacement tips to prevent pipettors from becoming cross-contaminated while pipetting samples that contain template. These types of pipettors and tips are available from several sources and can be purchased in sterilized packs. It is important to remember that barrier tips cannot be autoclaved.

Pre-PCR Activities

The definition of pre-PCR is the protocols and equipment required for the isolation of nucleic acid and the assembly of the reaction to amplify the samples. During the last 10 years, there has been much progress in developing devices that perform these activities in an automated fashion. Most PCR laboratories still perform these tasks using manual procedures. What is the minimum needed to equip a PCR laboratory for sample preparation, PCR reagent preparation, and PCR assay setup? Because most of the activities revolve around pipetting of liquids, these activities should be examined most closely; in particular, the manual pipettors and pipette tips. As discussed previously, positive displacement tips or barrier methods should be used to pipette the template into the reactions as the last step. There is a risk of creating aerosols in the preparation for RNA and DNA templates. If a large number of specimens of one type are processed on a routine basis, the laboratory may wish to treat this method with care and perform it in a hood or biosafety cabinet. Because of the effectiveness of ultraviolet light (UV) for amplicon control, use of UV inside the cabinet prior to sample preparation or PCR reagent preparation is advisable. Alternatively, any one of a number of small, benchtop-size cabinets that use UV irradiation can also be utilized. These are dedicated to PCR use and are large enough to contain several pipettors, racks, and some reagents.

Controlling Contamination by Carryover  

An amplification consisting of 25 cycles yields up to 103 copies/mL of the amplicon. A 0.1 μL aliquot of this mixture contains 10° amplicons. By comparison, there are only 1.4x 10 copies of a single-copy gene in a microgram of human DNA. As a result, such contamination results in a false-positive reaction. It is not surprising that the biggest problem associated with the successful use of PCR is this potential for specimen carryover. This carryover can be controlled through a number of different techniques, as follows:

Biochemical Sterilization Of Previously Amplified Material:

Commercial kits are now available (Carryover Prevention kit, Cetus Corp., Norwalk, CT) that cause the enzymatic degradation of previously amplified material. The methodology incorporates deoxyuridine triphosphate (dUTP) into the amplicon instead of deoxythymidine triphosphate (dTTP) into the amplicon instead of deoxythymidine triphosphate (UNG) is added to reaction material. UNG specifically digests any DNA sequence that contains uracil. A template that contains thymidine will not be affected. Use of this method eliminates any product generated during a prior amplification.

Sterilization Of DNA By Exposure To Ultraviolet Light:

The exposure of double-stranded DNA to ultraviolet (UV) light results in the formation of dimmers between adjacent thymidine residues. These altered bases are incapable of extension by the enzyme. Exposure to UV light does not affect the action of either enzymes or single-stranded primers. It is therefore possible to treat all reagent mixtures with UV light prior to the addition of template DNA in order to eliminate any contaminating DNA. It is also preferable to treat the work area with UV light.

Strict Adherence To Proper Laboratory Technique:

It is essential to prevent the introduction of previously amplified material into future reaction mixtures. To this end, the following suggestions have been made.

v Physically isolate PCR preparations and products: As separate clear room and biological safety cabinet are required for set - up of the reaction. Processing of amplified material must be performed in a totally different work area

v  Autoclave solution: Autoclaving degrades DNA into fragments of low molecular weight.

v  Aliquot reagents: Do not repeatedly open containers of buffers, enzymes, nucleotides, or primer stocks. Instead, prepare single - use aliquots and discard any remaining material after set-up of the reaction.

v  Use disposable gloves and change gloves often during set-up: DNA may splash onto gloves when the tops of microcentrifuge tubes are opened. Frequent changing of gloves reduces the possibility of transferring DNA between specimens. The powder that is used in latex gloves also interferes with the action of Taq DNA polymerase, It is essential to wash any excess powder off the outside of gloves prior to setting up a reaction.

v Avoid splashes: Perform a quick spin in a microcentrifuge before opening it, and pay close attention to careful pipetting techniques.

v Use positive-displacement pipettes or aerosol resistant tips on air-displacement pipettes: There are essential to prevent carryover. DNA contaminates the barrel of conventional air-displacement pipettes. The contaminating DNA is easily transferred to subsequent reactions.

v “Premix” reagents: In order to avoid excessive pipetting while setting up a reaction, prepare master mixes that contain all components except for the sample DNA.

v  Add DNA last: adding the DNA as the last step decreases the chance of carryover.

v Choose positive and negative controls carefully: Positive controls that contain large amounts of template DNA should be avoided, because their use would increase the likelihood of cross-contamination. Instead, use the lowest dilution’s that generates a positive result.

Sterilization of Reagents

Because PCR laboratories perform some molecular biology methods that require sterile reagents, some may need to be autoclaved. The single most critical reagent is water. Sterile USP water can be quickly converted to PCR water by filtering it through two 0.45-micron nitrocellulose filters. These filters have a very high binding capacity for nucleic acid and proteins. If the laboratory is involved in amplification of very small quantities of bacterial DNA, the USP water should be autoclaved separately from all other reagents before filtration. In general, reagents and solid items destined for the pre-PCR lab should be autoclaved separately from other supplies.

SUMMARY

Avoiding contamination is the most essential tool for the success of PCR laboratory operation. Here we have provided a brief overview of essential precautions that needs to be considered to avoid contamination in PCR laboratory operation.