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INTRODUCTION
One of the biggest strengths of PCR(e) for DNA typing is the degree to which DNA In a DNA-typing
can be amplified. Starting with a single DNA molecule, millions or billions of laboratory, every
DNA molecules can be synthesized after 32 cycles of amplification. This level of precaution must be
sensitivity allows scientists to extract and amplify DNA from minute or degraded taken to ensure that
samples and obtain useful DNA profiles. In this context, the sensitive nature of
PCR works in a lab's favor, but it can cause problems if great care is not taken
results are accurate
to avoid contaminating the reaction with exogenous DNA. Three main categories and unexpected
of exogenous DNA have the biggest impact on DNA-typing laboratories. These are: peaks or bands do
1) DNA from the analyst, 2) DNA from other samples in the lab, and 3) DNA not cast doubt on
fragments of the allelic ladder used to determine the size of amplified alleles. the data.
DNA from nonhuman sources, such as bacteria contaminating a casework sample,
will not be amplified and detected because STR systems are species-specific (1).
Here we discuss ways to detect DNA contamination and present tips to minimize
contamination problems.
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contamination is not always this
simple. The contaminating allelic ladder
Figure 1. An example of allelic ladder contamination in a PowerPlex 16 sample amplification.
may be from a different STR system. Sample peaks are evident but are masked by the amplified allelic ladder fragments, especially in the
If the primer sequences are identical smaller loci.
or the primers are nested, additional
peaks corresponding to alleles of loci cycles in the amplification program by buffer. Use autoclaved water, change
common to both systems appear. For 24 cycles (10/20 or 10/18 cycling). vials and wash the buffer reservoir as
example, if the GammaSTRMultiplex Alternatively, reduce the injection time a part of normal weekly maintenance
(Fluorescein) D16S539, D7S820, for capillary electrophoresis (CE) of the instrument.
D13S317, D5S818 Allelic Ladder instruments or the amount of Unexpected peaks can also be caused
Mix(b) is introduced into a negative amplification product loaded onto the by pull-up or bleedthrough. Pull-up can
control reaction assembled using the gel. Also, be sure that the samples are occur when a poor or incorrect matrix
PowerPlex16 System, many of the completely denatured by heating the has been applied to the samples.
alleles present in the GammaSTR samples for the recommended time If the peak heights are excessive,
Multiplex Ladder Mix will be detected and cooling on crushed ice immediately re-inject samples using a shorter
(Figure 2). prior to loading the gel or capillary. injection time or load less sample
CE-related artifacts (spikes), such on the gel. If the matrix or spectral
OTHER POSSIBLE EXPLANATIONS as those caused by minor voltage calibration is not performing optimally,
FOR UNEXPECTED PEAKS changes or urea crystals passing by generate a new matrix and apply it to
When checking for additional peaks, the laser, may also result in the samples (ABI PRISM310 and
be aware of other possible sources of unexpected peaks. Spikes sometimes 377 instruments), or perform a new
unexpected peaks that are not appear in one color but often are calibration and re-analyze the samples
indicative of DNA contamination. For easily identified by their presence in (ABI PRISM3100 and 3130
example, stutter peaks or n1 peak more than one color. Contaminants in instruments).
heights will be high if excessive the water used with the ABI PRISM
amounts of DNA template are used 310 Genetic Analyzer or other CE
or samples are overloaded. To remedy instruments may generate unexpected
this, use less DNA template (we peaks in the blue and green dye
recommend 0.51.0ng of template colors. This includes the water used
per reaction) or reduce the number of to dilute the 10X Genetic Analyzer
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CONTAMINATION
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with a dilute bleach solution.
Figure 2. Contamination of a no-template negative control PowerPlex 16 reaction with allelic
ladder from the GammaSTRMultiplex (Fluorescein) D16S539, D7S820, D13S317, D5S818. CONCLUSION
In a DNA-typing laboratory, every
RESOLUTION dropout. For equipment that cannot
precaution must be taken to ensure
If DNA contamination occurs, there are be treated with bleach, exposure to
that results are accurate and
steps that can be taken to eliminate ultraviolet light can eliminate DNA
unexpected peaks or bands do not
the contaminant. Individual solutions contaminants (24).
cast doubt on the data. In this article,
can be identified as contaminated Once the laboratory equipment has we showed examples of reactions
and discarded. To check solutions for been cleaned and any questionable contaminated with allelic ladder,
contamination, assemble negative reagents discarded, assemble a discussed methods to eliminate DNA
control reactions using new reagents number of reactions without DNA contaminants and offered tips to
known not to be contaminated, and template. Carefully examine the prevent DNA contamination in the
add one of the suspect solutions to amplification results looking for future. By diligently following these
each reaction. A reaction that shows additional peaks that can indicate recommendations, you can prevent
amplified products indicative of continued DNA contamination DNA contamination from ever
contamination is evidence that the problems. becoming a problem in your laboratory.
solution added was contaminated.
However, it is often easier, less time- PREVENTING CONTAMINATION REFERENCES
consuming and more reassuring to
One of the most efficient safeguards 1. Krenke, B. et al. (2002) Validation of a
simply discard all solutions that have 16-locus fluorescent multiplex system.
to prevent contamination is the
come into contact with samples that J. Forensic Sci. 47, 77385.
separation of pre- and postamplification 2. Sarkar, G. and Sommer, S. (1990) Shedding
demonstrate contamination.
areas and equipment. Use a dedicated light on PCR contamination. Nature 343, 27.
Wash all surfaces and rinse pipette set of pipettes, preferably with 3. Fox, J.C. et al. (1991) Eliminating PCR
barrels with a dilute bleach solution aerosol-resistant (barrier) tips, in the contamination: Is UV irradiation the answer?
J. Virol. Methods 33, 37582.
(2% to 3%). Be sure to rinse any pre-amplification area. Do not enter the
4. Ou, C.Y., Moore, J.L. and Schochetman, G.
equipment that comes into contact pre-amplification area after handling (1991) Use of UV irradiation to reduce false
with reagents well, since residual amplified samples or allelic ladder. positivity in polymerase chain reaction.
bleach can cause allele or locus Many analysts plan their day so that BioTechniques 10, 4426.
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