6 Responses to “The Week 95 Forensic Science Geek of the Week Challenge”

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  George Schiro, MS, F-ABC says:

  April 22, 2012 at 4:30 pm

  The concept pictured in the electropherograms is that low copy number (LCN) polymerase chain reaction (PCR) techniques may produce DNA types that do not accurately reflect the actual source of the DNA. LCN typically uses increased PCR cycle numbers on extremely low amounts of template DNA.

  This is from the discipline of forensic DNA analysis.

  The controversy surrounding it is that the LCN technique stretches the technology beyond the manufacturer’s recommended PCR cycle number and uses less template DNA than the manufacturer’s recommended amount. Some in the field believe that these stressors in the system will lead to inaccurate DNA results since these DNA kits were not designed and developmentally validated for LCN techniques. Others feel that with the proper internal validation studies and interpretation guidelines, LCN is an additional useful technique.

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  Christine Funk says:

  April 22, 2012 at 4:53 pm

  1. What sort of con­cept is pic­tured above?

  The phenomenon displayed, peak height imbalance, allelic drop out and in, and high stutter occur when low levels of DNA are subjected to 31 amplification cycles. DNA peak heights from a single source are expected to be approximately the same. They appear imbalanced with low levels of DNA input. Alleles also are known to ‘drop out’ (or disappear from detection) when testing low levels of DNA. Allelic drop in (I like to call them ‘Volunteer alleles’) can also show up when testing low levels of DNA. Finally, ‘stutter’ occurs during amplification when a true chunk of DNA ‘breathes’, bending outward and making a copy 4 base pairs less than the original. This copy is then replicated throughout the rest of the amplification process. Typically, stutter appears around 10 percent of the higher, true peak (give or take, depending on the locus involved). ‘High stutter’ is yet another stochastic effect which occurs when testing low levels of DNA.

  2. What foren­sic dis­ci­pline is this from?

  forensic DNA testing.

  3. Are there any known con­tro­ver­sies sur­round­ing it?

  Ummm, yeah. Since we can rely on these effects occurring, but cannot reliably determine when or in what form, interpreting low level DNA test results is subject to misinterpretation. The presence or absence of alleles can include or exclude possible contributors. When alleles drop out, or appear, or appear to be alleles when they are truly stutter, interpretation can be challenging at best.

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  Mehul B. Anjaria says:

  April 22, 2012 at 10:05 pm

  These are electropherograms associated with forensic DNA analysis. The results depicted here are from performing PCR on samples of DNA using the Identifiler kit followed by capillary electrophoresis. The electropherograms show ‘peaks’ representing instrumental detection of DNA. The peaks are sized, classified, and labeled by software so that the types detected can be compared to the DNA types of known DNA samples.

  The Identifiler PCR process involves making copies of 16 areas of the DNA that are used for typing and comparison and adding a fluorescent tag to these copies. In the normal course of business the PCR process is run for 28 cycles using approximately 1 nanogram (1,000 picograms (pg)) of DNA.

  The results shown here are for very low levels of DNA. A diploid human cell will have approximately 6 pg of DNA, so the results shown are for typing approximately 2-5 cells. This is referred to as ‘Low Copy Number’ (LCN) DNA testing. In an effort to boost the sensitivity of the testing, the PCR cycles have been increased from 28 cycles to 31 to create a greater number of copies of DNA for study.

  This is controversial because it goes outside of the bounds of the manufacturer’s recommendations and unexpected results occur routinely. LCN is best limited to careful use as an investigative lead due to the inherent issues. In fact, protocols for interpretation frequently call for at least 3 runs of the testing to determine which results are the most consistent within the 3 tests, and therefore more likely to be reliable.

  The results show issues that can occur with LCN typing. Essentially, you can lose information, lose the ability to judge whether a mixture is present, or even start seeing irreproducible results suggesting sporadic contamination. In short, it is difficult if not impossible to have strict interpretation guidelines for this sort of analysis since a range of expected outcomes is difficult to delineate.

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  Laura Magnuson says:

  April 22, 2012 at 10:27 pm

  Stochastic effects that randomly occur when PCR amplifying low amounts of DNA using an increased number of PCR cycles. The STR-typing kit, amount of DNA and number of PCR cycles along with the correct genotype for each example are listed at the bottom

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  Okorie Okorocha says:

  July 21, 2012 at 6:27 pm

  These are PCR anomalies from say using a low copy number, 31 cycles is
  high which usually means they had a very small sample.

  Its forensic dna analysis.

  The low copy number issue is huge. You get a lot of artifiacts, weird
  shapes, allelic drop outs and drop ins,

  With a low copy, you are very prone to contamination and multiplying.

  The graphs show low copy and/or contamination and this can cause
  incorrect matches, false positives, false negatives, or data that
  makes no sense.

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  Okorie Okorocha says:

  August 2, 2012 at 8:21 pm

  These are PCR anomalies from say using a low copy number, 31 cycles is
  high which usually means they had a very small sample.

  Its forensic dna analysis.

  The low copy number issue is huge.  You get a lot of artifiacts, weird
  shapes, allelic drop outs and drop ins,

  With a low copy, you are very prone to contamination and multiplying.

  The graphs show low copy and/or contamination and this can cause
  incorrect matches, false positives, false negatives, or data that
  makes no sense.