MiniFiler™ FAQS: Challenging Samples
Q: What are common misconceptions about handling inhibited/degraded samples?
A: Inhibited samples are often confused with degraded samples (and vice versa), and although the two types of samples present difficulty to the analyst, they can often be distinguished after close examination of the quantification results, DNA profiles and a consideration of the sample's origin. The Quantifiler® Human DNA Quantification Kit contains an internal PCR control (IPC) that can serve as a useful indicator of potential inhibition.
Q: What information about the sample may assist in identifying challenging samples?
A: Careful consideration should be given to the totality of the information regarding a sample:
- What is its origin, if known? Whole blood sample, bone, buccal swab, black denim, etc.
- What type of substrate was it extracted from? Cotton tipped applicator, dyed fabric, leather, plant material, etc.
- What natural elements has this sample been exposed to? Heat, sunlight, moisture/humidity, etc.
- Does the sample contain possible inhibitors of DNA polymerase such as textile dyes, heme or humic acid?
Q: How do you tell the difference between inhibited and degraded samples?
A: The presence of inhibitors that co-extract with sample DNA may be detected using the Quantifiler® Human DNA Kit through an evaluation of the IPC results. Inhibited samples exhibit an IPC with a higher than expected Ct value or an IPC that does not produce a signal which crosses the threshold. Samples exhibiting potential inhibition may result in lower than expected quantification results requiring further sample clean-up or dilution in order to obtain accurate quantification. Neglecting to re-quantitate an inhibited sample after clean-up or dilution may result in off-scale data for smaller fragments when run on a Capillary Electrophoresis instrument. A degraded sample (with no inhibitors) would produce an expected and consistent IPC value. Quantification of degraded DNA may result in an overestimate of the amount of DNA present with preferential amplification of smaller amplicons relative to the larger amplicons. Addition of higher than normal amounts of input DNA may assist in recovery of information from the longer amplicons.
Q: How can extraction/purification methodology impact results from compromised samples?
A: The presence of PCR inhibitors that are co-extracted with sample DNA can result in allele dropout or imbalances in heterozygous loci. A number of techniques exist to overcome inhibition of amplification including:
- Use of extraction methods which effectively remove inhibitors from the sample
- Dilution of extracted DNA which consequently dilutes the inhibitor, thereby minimizing the effect
- Addition of BSA to the PCR reaction
Q: What indicators of degradation and/or inhibition should I look for in sample data?
A: In such samples, the ability to amplify the longer amplicons is diminished relative to the ability to amplify the shorter amplicons. Samples containing inhibitors may exhibit partial profiles similar to those generated from degraded samples, resulting in complete loss of all alleles or resulting in drop-out or imbalance at select loci. Until recently, the ability to recover information from degraded samples was limited. Technological improvements have been implemented in the AmpFℓSTR® MiniFiler™ PCR Amplification Kit that include moving the primers in closer to the locus repeat regions. This has allowed the generation of smaller amplicons, thus increasing the probability of obtaining a full profile from a degraded sample.
Q: What is the best strategy for recovering information from degraded and/or inhibited samples?
A: The MiniFiler™ PCR Amplification Kit has been demonstrated to yield the greatest amount of information from samples that have previously produced partial profiles or no profile at all using existing commercially available autosomal amplification kits. In the presence of PCR inhibitors, the MiniFiler™ Kit outperforms other commercially available multiplex kits with regard to recovery of genetic information. The MiniFiler™ Kit contains 8 autosomal markers and the sex-determining marker amelogenin. Use of a dual-amplification strategy using MiniFiler™ and Identifiler™ Kits is the best strategy for recovery of all 15 autosomal markers from compromised samples.