Thursday, September 11, 2014

Challenging DNA

[Thanks to Tom Bartee for this post]

Now that forensic DNA has been used to putatively identify Aaron Kosminski as Jack the Ripper, this may be a good time to consider some of the possible problems with such evidence. We will likely be seeing more use of DNA from “touch” sources on guns or drug packaging, which raises the issue of “low copy number” DNA. I hope that this will help you recognize some issues arising from the forensice use of LCN DNA.

What is LCN?

LCN involves a small amount of DNA template available for PCR amplification, typically said to less than 100 picograms (PG), although some analysts consider the cutoff to be at 200 pg. A picogram is one-trillionth of a gram. Because a typical human cell weighs about 3 pg, LCN analysis may involve the DNA from just a few cells.

When is LCN an issue?

LCN is common with “touch” DNA, i.e., DNA obtained from cells left in fingerprint residues, saliva, sweat, or sebum. Such small amounts of DNA may be left on an item through “secondary transfer,” when DNA from one person is first transferred to another person (like during a handshake) or to an item, and that DNA is in turn transferred to the evidence. LCN is also frequently a concern in mixed samples with a minor contributor. Even if the total amount of DNA amplified is 1 ng, the minor contributor profile in a mixed sample with one part minor contributor to nine parts major contributor could be LCN. The crime lab analyst may fail to understand that it is the amount of DNA of each contributor that is important, rather than the gross amount of DNA in a mixed sample.

How will I know whether LCN is an issue?

You cannot assume that the lab report will disclose that LCN DNA has been analyzed. In fact, some labs will engage in LCN analysis but deny that it is actually LCN. You will probably need an expert to know with certainty, but LCN is more commonly encountered in cases involving mixtures, partial profiles, or unusually high random match probabilities (1 in 10 thousand rather than 1 in 10 trillion).

How do labs react to LCN?

Labs may increase the number of amplification cycles, from the standard 28 cycles to say 34, radically increasing the amount of DNA available for testing, but also creating dangers discussed below. Rather than increasing cycles, labs can reduce the volume of the PCR product, thereby increasing the concentration of DNA in it, or they may added more amplified product than normally used.

What are the dangers of LCN analysis?

The immediate problems associated with LCN arise from the fact that the small amounts of template DNA may be below the “stochastic threshold,” i.e., the point at which random factors may influence the testing more than does DNA from the sample. The PCR technique uses a series of PCR cycles to amplify a tiny amount of DNA, so any random effects present at the beginning of the process will also be amplified. Resulting “stochastic effects” may include:

“allele drop-in,” in which alleles not present in the sample DNA appears in the amplified DNA from a source of contamination – with such a small amount of sample DNA present, it may be overwhelmed in the amplification process extraneous DNA (from the lab or from innocent third parties)

“heterozygote peak imbalance” resulting from the preferential amplification of one of the two alleles at a locus, resulting in peak height imbalance or even allelic drop-out, in which one of the alleles fails to amplify

“stutter peaks,” which are an artifact of the PCR amplification process, may become larger than are normal with high copy number DNA, resulting in the analyst calling an allele that is absent from the crime scene sample DNA
Another problem associated with LCN analysis is consumptive testing, i.e., not leaving DNA for independent retesting.

What precautions should labs take when handling LCN?

LCN DNA from an evidence sample may be overwhelmed by DNA from contamination. Labs should insure sterility in the lab, requiring lab personnel to wear masks, gloves, and gowns, and devices and lab benches should be sterilized with bleach. The profiles obtained from LCN testing should be compared to the staff elimination database.

What should I do if I have a LCN case?

Get an expert, of course. You may have a good Daubert challenge to the evidence, as the lab’s validation studies and the lab equipment user’s manuals may not support the procedure used by the lab in handling the analysis, e.g., if the lab increased the number of PCR cycles.

A good DNA resource for defense lawyers is DNA for the Defense Bar, published by the DOJ’s National Institute of Justice (June 2012), available on-line at:

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