Prevention of carry-over

1. Introduction
2. The solution to a contamination
3. Prevention of carry-over by physical separation of preparation and
analysis
4. 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. Introduction
One 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 l. 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 problem
If 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
analysis
A 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-over
The 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 irradiation
Direct 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). Isopsoralen
Incorporation 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 method
Incorporation 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. Conclusion
Carry-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 UracilDNA 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 uracilcontaining 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 (95C) 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 postPCR 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.