Prevention of carry-over

1. Introduction 2. The solution to a contamination 3. Prevention of carry-over by physical separation of preparation and

analysis4. Whole in One and NucleoLink Tape 8 decreases carry-over 5. Prevention of carry-over by UV-irradiation

a. Prevention of carry-over by changing the product composition from the template   Isopsoralen

b. The UNG method 6. Conclusion

1. IntroductionOne of the major problems in the use of PCR as an analytical tool is the risk of having new reactions contaminated with old, amplified products. When this happens, all reactions will be positive, and it is not possible to distinguish between amplified products from the contamination or a true, positive sample. In addition to taking precautions to avoid or control this carry-over of old products, it is necessary to include a blank reference reaction in every PCR experiment to check for carry-over (  ). In order to be certain that all results are reliable, there must be no amplified products after the temperature cycling (  ). A carry-over contamination will be visible on the agarose gel as faint bands (  ) and may be observed in the DIAPOPS analysis as an increase in the background signals (  ). Furthermore, it is also very important to include a positive sample. If, contrary to expectation, the sample is negative, none of the results can be considered as trustworthy.

It is conceivable that the reagents used to prepare the PCR may be contaminated. After the amplification a positive sample may contain 250 ng PCR product in 50 µl. This gives a total of 3.9 1011 copies of a 600 bp double-stranded product. One thousandth of a microlitre of this reaction will contain approximately 8 million copies. If a very small and invisible aerosol is formed when the PCR vessel is opened, there is a possibility that this aerosol can contain a very large number of amplified products. Furthermore, the microscopic droplets in an aerosol are able to float for a long time in the air, and if there is turbulence in the room, they can be carried a long way. Considering the fact that only one copy is enough to create a false positive reaction, it is obvious that great care must be taken to avoid this carry-over contamination.

2. The solution to a contamination problemIf the reagents used for PCR mix become contaminated, there is only one good way to eliminate this contamination. This is by replacing ALL reagents and stock buffers with new chemicals and new water which have never been in contact with the areas of sample preparation and PCR analysis.

There is no point in looking for the one reagent which is contaminated. Contamination may occur in more than one solution, or it can be in a stock solution where it may be difficult to find the source. The time wasted in this search exceeds the costs of exchanging all the reagents.

3. Prevention of carry-over by physical separation of preparation and analysisA very efficient way of preventing carry-over is to physically divide the area of reagent mixing and sample preparation from the area of product analysis (Kwok & Higuchi, 1989). This separation should be considered very seriously, and no equipment or reagent which has been placed in the room for PCR product analysis should ever be transferred back to the room where the sample and PCR reagent are prepared. There must be specific equipment in both rooms. If these physical precautions are taken, most problems can be prevented.

4. Whole in One and NucleoLink Tape 8 decreases carry-overThe NucleoLink Strips are closed with a heat-stable tape (Nunc Tape 8, Cat. No. 249719). After amplification, this tape ensures that there is no difference in pressure between the inside of the NucleoLink well and the surroundings. For this reason the formation of aerosols is diminished, and the risk of carry-over contamination is reduced. Furthermore, the analysis is made on the solid phase PCR products and takes place in the same well. This approach is called Whole in One. If the liquid phase is discarded and care is taken not to contaminate the laboratory, there should be only a small risk of contamination when performing the DIAPOPS in NucleoLink Strips (  ). However, it is still very important to maintain the physical compartmentalization of the sample preparation area and the analysis area.

In addition to taking these precautions in the laboratory, that is using separate locations for mixing and product detection, several procedures can be used for destruction or alteration of the contaminating old PCR product.

5. Prevention of carry-over by UV irradiationDirect UV irradiation can effectively remove contaminating DNA (Rys & Persing, 1993 and Sarker & Sommer, 1990), but the irradiation of the PCR reagents must take place before addition of polymerase, primers, and template DNA. The subsequent addition of these substances to the Strip can cause a new contamination (Erlich   et al.   1991 ). Furthermore, this approach may be inefficient because the large numbers of mononucleotides present in the reaction will absorb much of the UV light (Frothingham   et al.   1992 ).

6. Prevention of carry-over by changing the product composition from the template

A further approach could be to make the DNA composition of the PCR product different from the natural template DNA composition. This altered composition is intended to make the PCR products sensitive to treatment that will not alter the template DNA. The treatment of the closed PCR vessel just before amplification should make the contaminating PCR product unable to participate in the amplification.

6. a). IsopsoralenIncorporation of a photochemical reagent (isopsoralen) into the product during amplification will create a difference in composition between the template DNA and the amplified PCR products (Rys & Persing, 1993). Light treatment of the closed PCR vessel will render previously formed PCR products unable to act as templates for further amplification. The hybridization abilities of the product are not changed, but the detection capabilities on agarose gel can be decreased due to reduced binding of EtBr. This approach for carry-over control has not been tested with DIAPOPS, but since this method does not change the hybridization ability of the product, it should be adequate in DIAPOPS.

6. b) The UNG methodIncorporation of dUTP into the amplified fragments will also alter the composition of the product so that it is different from the template DNA composition (Longo   et al.   1990 ). The enzyme Uracil-N-Glycosylase (UNG) is added together with the normal PCR enzyme to the reaction mix. The UNG enzyme will cleave the uracil base from DNA strands before amplification, and leave all the old amplified products unable to act as templates for new amplification, but will not react on unincorporated dUTP or new template. This will efficiently remove contaminating PCR products from the reaction after the PCR vessel has been closed, and thus no new contamination is possible.

However, the use of dUTP in PCR reactions to prevent carry-over can cause problems when the products are used in a later hybridization study, due to the low capability of uracil to act in hybridization (Carmody & Vary, 1993). dUTP is incorporated instead of dTTP. When a probe rich in T's is amplified with the substitution of dTTP for dUTP in the reaction mixture, a later hybridization signal with the probe may be eliminated. To avoid the decrease in hybridization signal the probe binding site should be chosen with no more than 25% T's, and without stretches of poly-T. Furthermore, the PCR should contain equal concentrations of dUTP and dTTP and not only dUTP. In contrast to the decrease in hybridization signal is the increase in product amplification when using dUTP, especially when AT-rich target sequences are selected. This is probably because the incorporation of dUTP decreases re-annealing of formed PCR products which would prevent primers from annealing. If this approach is used to increase the product yield, the primer binding sites should be selected with a low content of T's, since primer annealing also will be inhibited by dUTP

incorporation (Carmody & Vary, 1993). The dUTP method for carry-over control has not been tested in DIAPOPS, but if care is taken in the design of the probe and primer sequences to avoid T's, the method is expected to be very efficient in DIAPOPS.

7. ConclusionCarry-over of old amplified PCR products can be a very serious risk to the DIAPOPS analysis. The best way to prevent this contamination is to physically divide the PCR working areas. Furthermore, actions such as UV irradiation of PCR mix and incorporation of reagents into the newly formed PCR product can be used to alter it from the template. However, these methods have different drawbacks, and have not been tested in DIAPOPS. Whole in One, and the NucleoLink Tape 8 decrease the risk of carry-over contamination in the DIAPOPS procedure.

Preventing Carry-over Contamination with Uracil-DNA Glycosylase

The polymerase chain reaction (PCR) can amplify a single molecule over a billionfold. Thus, even

minuscule amounts of a contaminant can be amplified and lead to a false positive result. Such

contaminants are often products from previous PCR amplifications (carry-over contamination).

Therefore, researchers have developed methods to avoid such contamination.

One common strategy is substituting dUTP for dTTP during PCR amplification, to produce uracil-

containing DNA (U-DNA). Treating subsequent PCR reaction mixtures with Uracil-DNA

Glycosylase (UNG) prior to PCR amplification and subsequent cleavage of apyriminic

polynucleotides at elevated temperature (95°C) under alkaline conditions (during the initial

denaturation step) will remove contaminating U-DNA from the sample (see figure below). This

method, of course, requires that all PCR-reactions in the lab have to be carried out with dUTP

instead of dTTP.

Note the following when using dU-containing PCR products in downstream applications:PCR products containing dU perform as well as those containing dT when used as

hybridization targets or as templates for dideoxy sequencing.PCR products containing dU can be cloned directly, if they are transformed into ung–

bacterial hosts.A dU-containing substrate is readily digested by some common restriction enzymes (e.g.

Eco RI and Bam HI), while others show reduced activity (e.g. Hpa I, Hind II, Hind III) on these substrates.

We do not recommend the use of dU-containing DNA for protein binding or DNA-protein interaction studies.

The following Roche Applied Science products are suited for preventing carry-over

contamination in PCR:

The first thing you must consider when you set up your PCR laboratory is the potential risk of carry-over contamination of amplified products. PCR is such a powerful technique that even a few molecules of template DNA can be amplified to billions of copies in a single reaction. Thus, amplified products that can be transferred from previous amplifications always represent a potential contaminant to successive amplifications (carry-over contamination). To reduce the risk of carry-over contamination, there are some steps you can take:

Separate DNA extraction, pre-PCR set up and post-PCR examination facilities. (Preferably into three different rooms). Do not move equipment like pipettes, racks, microfuges etc. between facilities. 

Always include a negative (no DNA) control in your DNA extraction and PCR set ups. 

Each person in the lab should have his/her own set of pipettes and reagents for DNA extraction and for pre-PCR. Reagents should be made up and stored in small aliquots that can be discarded if carry-over contamination is suspected or observed. 

Always treat tubes and solutions post-PCR assuming that amplified products are present. Use separate post-PCR pipettes for this work. 

Avoid creating aerosols. Aerosols are easily created when pipette tips are ejected, or if pipettes are waved vigorously. 

Use filter tips to reduce the risk of transferring DNA between tubes. 

If possible, have UV-lamps decontaminate the laboratory whenever people are not present. 

If you are performing nested PCR, you need to set up separate pre- and post-PCR environments and routines for this work.