Emerging and available DNA preparation methods designed for forensic and other sample types help to remove inhibitors and improve PCR efficiency.
Polymerase chain reaction (PCR) is a very common technique used in many fields including biological research, forensics, and drug discovery. A major weakness of PCR, in general, is that the polymerase enzyme used to catalyze the reaction is very sensitive to the presence of inhibitors in the reaction mixture. Frequently, inhibitory substances such as magnesium, bovine serum albumin, humic acid, and indigo dye, present in the sample containing the DNA template, are often the result of improper or incomplete DNA purification. Forensic samples are especially notorious for containing numerous PCR inhibitors.
But don’t take my word for it. Susan Greenspoon, PhD, forensic molecular biologist, works at a case-working and forensic DNA analysis lab at the Virginia Department of Forensic Science in Richmond, Va. She says that “forensic samples are typically very dirty because they might be collected from a floor or an old shoe or from dirt from the ground. You could have lots of materials that have been deposited right on the dirty ground that, by nature of the forensic science and the kinds of scenarios in which these samples are produced, are frequently full of inhibitors.”
In preparation for short tandem repeat (STR) analysis, forensic examiners purify genomic DNA from samples using a robotics-operated, guanidinium-based, solid-phase extraction protocol, specifically designed for such samples. The typical sample types analyzed in the lab include blood, saliva, swabbing from a gun, clothing, etc. “The solid-phase extraction is often an excellent way to remove inhibitors because they don’t usually co-purify. Usually, the nucleic acid sticks with high affinity to the silica,” says Greenspoon. But typical inhibitors found in forensic samples such as humic acid simply wash away using this method.
In most cases, Greenspoon uses AmpliTaq Gold (Applied Biosystems, Foster City, Calif.) to perform human identification from forensic samples. And although she has had great success using this method, she does face the occasional inhibition in her PCR reactions and attributes it to the robotic extraction procedure she uses for isolation of genomic DNA. “We usually just overcome that by diluting the sample a little bit to dilute the inhibitors. It dilutes the DNA concentration going into the amplification cocktail, but it also dilutes the inhibitors. That is usually good enough to get beyond the PCR inhibition,” says Greenspoon.
Washing away inhibitors
QIAGEN produces DNA purification kits that operate on the principle of specific binding of DNA to silica surfaces in the presence of chaotropic salts. The silica is attached to either a plastic spin column or a magnetic bead (see The MagAttract Procedure). Using this method, nucleic acid is purified but residual contaminants bound to the silica are washed away using low and high salt buffer washes. “For most sample types, this procedure will remove potential inhibitors completely. Some sample types, however, need specific treatments, because they contain huge amounts of inhibitory substances and/or they contain substances showing binding characteristics similar to DNA, making it hard to separate analyte from inhibitor,” says Mario Scherer, PhD, scientist in the Department of Nucleic Acid Preparation Research, QIAGEN GmbH, Hilden, Germany. Scherer cites some examples:
• Various substances found in stool or soil samples can inhibit PCR; treatment of the lysate with InhibitEX tablets can remove inhibitors prior to PCR.
• Bone and teeth contain high levels of calcium, which can inhibit PCR; calcium can be removed by treating the sample with EDTA-containing buffers prior to PCR.
Scherer also points out that because samples taken from crime scenes often contain minute quantities of DNA, all steps in the protocol provided in DNA preparation kits must be as efficient as possible. And this includes efficient lysis conditions, the use of proteinase K in denaturation buffers to reduce PCR inhibition by proteins in the sample, and the use of carrier RNA to improve recovery of trace amounts of DNA from the sample.
“[Qiagen] will continue to develop optimized manual and automated kits dedicated to forensic samples,” says Scherer.
Let’s get physical
Andre Marziali, PhD, P.Eng., director of engineering physics, University of British Columbia, Vancouver, B.C., Canada, has worked with forensic samples collected by the Royal Canadian Mounted Police (RCMP). Although Marziali and his colleagues have not worked with actual casework samples, they are using forensic samples (as well as other sample types) to develop a technology that eliminates PCR inhibitors from those samples. “The forensic samples the RCMP provided us were those that they felt were challenging to their own prep methods,” says Marziali. “So we used them to test our technology, which worked very well. They all ended up amplifying after our purification.” Marziali and his colleagues have tested this new technology on forensic samples such as swabs containing blood and soil (the inhibitor here is the humic acid found in soil); and blood-stained denim (the inhibitor here is the indigo dye found in denim).
In his research lab, Marziali and his colleagues have spent the last four years developing this novel, electrophoretic technology to extract DNA from difficult samples, such as forensic samples. Their technology has been licensed into Boreal Genomics, North Vancouver, B.C., Canada, a new company formed to commercialize this technology, and already have several prototype instruments out in the field. The instrument works through a novel electrophoretic principle that separates DNA from contaminants such as humic acid and indigo dye. In this method, the nucleic acid is isolated based on its physical properties, primarily its ability to be reshaped when traveling through an electric field. This is in great contrast to the way most DNA purification methods work, which are largely based on DNA’s chemical properties (e.g., the ability of DNA to bind to silica). “It is the conformational entropy of the molecule that allows you to separate it from other molecules like humic acid that might have similar chemical characteristics but have different physical characteristics,” says Marziali. In head-to-head comparisons in which Marziali started with well-controlled mixtures of DNA and humic acid, and then through a series of dilutions he added increasing amounts of humic acid into the sample, he observed that this new electrophoretic technology was 100- to 1000-fold more effective at removing humic acids than conventional column and bead technologies. Effectiveness was measured by real-time PCR.
Other than inhibition …
A major challenge the forensic scientist faces is to perform accurate DNA profiling from mixed samples, i.e., samples in which there is DNA from more than one person. In this case, one source might be the actual perpetrator of a crime and the other might be the victim or someone unrelated to the crime. The underlying reason for this challenge is that it can be difficult to discern if alleles of a mixture came from the same genomic DNA template or from different templates. And having multiple sources of DNA can produce PCR artifacts, such as allelic drop-out, making the mixture that much more challenging to resolve.
Another issue forensic scientists must contend with is DNA degradation from environmental exposition of forensic samples. However, Scherer points out that Qiagen’s kits for DNA prep from forensic samples also provide stringent binding conditions to allow for recovery of DNA fragments as small as 100-bp in size.
Inhibition and other DNA-related sample preparation issues plague the forensic scientist, making sample analysis challenging. Luckily, there are kits and methods available to circumvent these issues, for the most part.
This article was published in Drug Discovery & Development magazine: Vol. 10, No. 9, September, 2008, pp. 22-24.
Filed Under: Genomics/Proteomics