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Stochastic Effects of LCN DNA Analysis

Home > Other DNA Markers & Technologies > Other Nuclear DNA Markers and Technologies > Low Copy Number DNA > Stochastic Effects of LCN DNA Analysis
The PCR Process

Due to the kinetics of the PCR process a small number of starting genomic DNA templates will result in the production of stochastic sampling artifacts.17 Thus the bimolecular reaction comprising a primer binding to its cognate template will be a random and rare event in the LCN milieu in which only a few target templates are available. Specifically, the molecular collisions required may or may not occur for each allele during each of the first few cycles resulting in a significant imbalance between alleles or, in extreme cases, total loss of one or both alleles.19 Only those alleles that are amplified efficiently during the first few cycles will be able to produce sufficient product to surpass the detection threshold of the standard PCR endpoint analysis.

DNA Profile Morphology

Depending upon the sample and the particular amplification, stochastically-induced allelic imbalance and drop out of one or both alleles will be commonly observed in LCN profiles (see figure below). Stutter peak height ratios are also raised in some LCN samples. Allelic imbalance and increased stutter complicate mixture interpretation and single allelic drop out produces false homozygous profiles. Locus drop out due to loss of both alleles, while regrettable, does not create novel profile interpretation issues.

Contamination and Spurious Alleles

The increase in sensitivity of LCN analysis permits the detection of low levels of extraneous DNA contamination that, while often present, is not normally seen with standard 28 cycle STR analysis.17 Thus alleles may show up in the profile that do not originate from the principal DNA donor(s) (see Figure) and, in control experiments, have been shown to occur with known single donor samples. Such allelic drop-in is more-often-than-not of unknown origin but could be due to DNA from a variety of intra-laboratory sources including consumable items and personnel. As a result LCN analysis should only be conducted in sterile laboratory facilities that have in place suitable engineering controls, akin to those employed for mtDNA analysis.

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NOTE TO USERS: The President’s DNA Initiative DNA Analyst Training program and assessment were completed and published in 2005, in cooperation with the National Institute of Justice. The science and techniques in the program are sound and proven, however, program content has not been updated to include tools and technologies developed and in use after 2005, including many kits and robots. Assessment questions address only content delivered in this program and may not contain the full range of tools in use in your laboratory.