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Repeated testing of replicated aliquots of the same diluted control DNA samples permits an evaluation of result reproducibility. This article will examine some simple validation experiments performed with low amounts of DNA and their implications. As forensic DNA analysts attempt to recover information from low amounts of DNA present in evidentiary samples, they will encounter stochastic or random sampling effects that occur in the early cycles of PCR amplification.
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When a limited number of DNA target molecules exist in a sample, the PCR primers used to amplify a specific region may not consistently find and hybridize to the entire set of DNA molecules present in the amplification reaction. With a heterozygous locus, where two alleles are present, unequal sampling of the alleles can result in failure to detect one or both of the alleles. Stochastic random variation is a fundamental physical law of the PCR amplification process when examining low amounts of DNA.
Stochastic effects are manifest as a fluctuation of results between replicate analyses. In other words, amplifying the same DNA extract twice can result in different alleles being detected at a locus. Since stochastic effects cannot be avoided when testing small quantities of DNA, there are essentially two schools of thought on how to handle these types of samples: Those who advocate the second approach usually enhance their method sensitivity, such as increasing the number of PCR cycles, to get as much out of the limited sample as possible.
For example, if the total amount of measured DNA is below pg, a laboratory may decide not to proceed with PCR amplification, assuming that allelic drop-out due to stochastic effects is a very real possibility.
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Alternatively, a laboratory may proceed with testing a low-level DNA sample, then evaluate the peak height signals and peak height ratios at heterozygous loci. Since the advent of quantitative PCR qPCR assays, DNA quantitation tests have become more sensitive—enabling quantities as small as a few genomic copies to be detected. However, it is important to keep in mind that qPCR also is subject to stochastic variation, especially on the low end of DNA quantity measurement. In an early paper discussing stochastic effects and limitations of PCR assays, Walsh et al.
Below roughly that amount, allele and locus drop-out would be expected and partial DNA profiles would result.
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Improved sensitivity in a detection technique is usually a valuable asset to enable results to be obtained from limited biological evidence. In fact, DNA testing has been applied successfully down to the single cell level 5. A more recent approach to high-sensitivity DNA testing uses only a three-cycle signal enhancement to provide a theoretical fold improvement in sensitivity 8. When pushing assay sensitivity through an increased number of PCR cycles, stochastic effects can become more evident.
For further information, see www. Likewise, the gain of signal with the high stutter or allelic drop-in could make a true single-source sample appear to be a mixture. Thus, when using enhanced interrogation techniques, such as a higher number of PCR cycles, further testing measures are required to avoid reporting incorrect results. To avoid or limit the possibility of getting the wrong answer when testing low amounts of DNA, replicate PCR amplifications are performed and a consensus profile developed 9.
Alleles that occur more than once i. Based on observations during validation studies, another layer of interpretation may be applied as well before the final consensus profile is reported. For example, specific loci, such as those larger in size, may exhibit a higher rate of allelic drop-out. When reporting results from these loci, a wildcard designation may be used in conjunction with a repeated single allele to account for potential allelic drop-out e.
However, amplification results from a single test can be unreliable due to allelic drop-out or allelic drop-in as noted earlier. Comparison of approaches when examining low amounts of DNA. Replicate amplification with development of consensus profiles and application of interpretation rules based on validation experience can lead to reliable results.
https://kingvenholaju.tk The red arrows indicate positions of allele drop-out, and the blue arrows indicate where severe peak height imbalance was observed. These electropherograms are available for review as pdf files at: Thus, boosting the cycle number did improve the sensitivity and overall success rate. The potential occurrence of allele drop-in shows the importance of replicate amplifications and development of consensus profiles to avoid miscalls when utilizing enhanced interrogation techniques. Green squares indicate that the full correct type was observed for that locus and PCR replicate.
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Yellow squares denote allele drop-out where one of the expected alleles is missing. Red squares highlight locus drop-out where both expected alleles are missing. Black squares indicate allele drop-in where an incorrect allele is observed above a 50RFU analytical threshold.
Thus, each STR-typing kit will perform differently and needs to be internally validated for the specific conditions being examined in your laboratory. Certain loci are more prone to allele and locus drop-out, depending on the STR kit used. In all cases, replicate testing and development of consensus profiles would have successfully excluded any incorrect calls due to allelic drop-in with the single-source samples examined in our study. Equally important is that across any group of three replicates, there was never an instance of an incorrect allele being called when two of the three replicates matched.
This web site, which is available at: These presentations are provided as pdf files, enabling easy access with Adobe Acrobat Reader. Two DNA samples that are heterozygous at all STR loci examined provide an opportunity to monitor peak imbalance and allelic drop-out under different conditions.
These data help illustrate the stochastic variation observed when amplifying low amounts of DNA, including allele drop-out, allele drop-in, high stutter and heterozygote peak imbalance. We encourage laboratories to submit their validation data for inclusion on this community resource as suggested previously The articles are listed according to four categories in order to reflect their relative reliability in scientific terms: In science, as in other fields, not all information is equally authoritative or helpful. Thus, the literature on low template DNA analysis is broken into several categories on the STRBase web site to reflect the variation in scrutiny and support.
Every lab faces samples with low amounts of DNA. If such an approach is taken, validation studies need to be performed to develop appropriate interpretation guidelines and to assess the degree of variation that can be expected when analyzing low amounts of DNA. Deciding where to stop testing or interpreting data can be challenging. Some laboratories stop testing based on a certain amount of input DNA, using validation data to underpin a quantitation threshold.
Others set stochastic thresholds that are used during data interpretation to decide what STR-typing data are reliable i. Performing experiments similar to those described in this article can help determine an appropriate stochastic threshold. With greater power to get results comes greater responsibility to report reliable results. Careful validation studies and development of appropriate interpretation guidelines will continue to be essential as the forensic DNA community moves forward with caution and care in analysis of low amounts of DNA.
Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U. Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.
Elsevier Academic Press, San Diego. Promega Corporation Web site. Accessed Month Day, Year. Contribution of an article to Profiles in DNA does not constitute an endorsement of Promega products. Products may be covered by pending or issued patents or may have certain limitations.
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