This article presents answers to a number of frequently asked questions about forensic casework sample processing on the Maxwell® 16 Instrument.
Q: What was the technical rationale for changing the extraction buffer used to preprocess samples prior to DNA isolation using the DNA IQ™ Casework Pro Kit for Maxwell® 16?
DNA yield from a forensic sample is a composite effect of two separate processes: extraction of a biological sample from a substrate and isolation of DNA from the extracted material. Screening a large number of sample types identified several samples (primarily panels of commercially available fabrics) that exhibit a much lower DNA yield, which was attributed to extraction using the Tissue and Hair Extraction Kit (Cat.# DC6740). Further investigation revealed a single reagent that restored the expected yield from these sample types
. In this way, the success rate of extraction was improved for a broad panel of sample types. Thus, the Casework Extraction Kit can be used in lieu of Tissue and Hair Extraction Kit for the majority of casework sample types, including many fabrics and swabs. However, there continues to be specific materials that may negatively affect the performance of DNA IQ™ chemistry
. As an added benefit, I noticed that the new reagent routinely improved the yield for the overwhelming majority of sample types tested, as measured by DNA quantitation using the Plexor® HY System and signal intensity in downstream STR amplification.
Q: What changes were made to the sample preprocessing conditions with the new Casework Extraction Kit?
With the introduction of the Casework Extraction Kit (Cat.# DC6745), there are three changes that were made to the extraction of samples from solid supports.
- The recommended incubation temperature was lowered to 56°C. The Casework Extraction Buffer extracts samples efficiently and does not require higher temperatures to be effective.
- The samples are now incubated in 400µl of Casework Extraction Buffer, 1-Thioglycerol and Proteinase K, as described in the DNA IQ™ Casework Pro Kit for Maxwell® 16 Technical Manual #TM332. This change was made to improve extraction efficiency (Figure 1). Subsequent experiments demonstrated no increase in yield using volumes higher than 400µl.
- An alternative reducing agent to dithiothreitol (DTT) is used: 1-Thioglycerol. 1-Thioglycerol is as effective as DTT at lysing sperm cells but does not require storage and shipping at –20°C. This allows the user to store 1-Thioglycerol at 2–10°C upon receipt and does not require thawing (as does DTT) prior to use.
Figure 1. DNA yield versus extraction volume for the DNA IQ™ Casework Pro Kit for Maxwell® 16.
One microliter of blood was dried on a panel of swabs overnight, then extracted in various volumes of Extraction Buffer, as indicated. After extraction, the total volume of Extraction Buffer was brought up to 400µl to ensure constant conditions in the subsequent DNA-binding steps, the solid support was removed using a DNA IQ™ Spin Basket, 200µl of Lysis Buffer was added and the sample lysate was added to well 1 of the DNA IQ™ Casework Pro cartridge. DNA from the resulting eluates was quantified using Plexor® HY System to estimate DNA yield. N = 8 for each treatment set.
Q: Will the new Maxwell® 16 High Strength LEV Magnetic Rod and Plunger Bar Adaptor improve DNA yield when using the DNA IQ™ Casework Pro Kit for Maxwell® 16?
The Maxwell® 16 High Strength LEV Magnetic Rod and Plunger Bar Adaptor (Cat.# SP1070) reduces residual resin in the LEV Cartridge and efficiently removes resin from eluted DNA at the end of each run. Research performed at Promega has demonstrated no improvement in DNA yield with the DNA IQ™ Casework Pro Kit for Maxwell® 16 in combination with the Maxwell® 16 High Strength LEV Magnetic Rod and Plunger Bar Adaptor. Multiple experiments were performed to support this conclusion.
Q: For the DNA IQ™ chemistry, I thought that the DNA IQ™ Lysis Buffer must constitute two-thirds of the volume at the binding step. Will my samples continue to bind efficiently with the new sample preprocessing recommendations?
The previous recommendation was that the DNA IQ™ Lysis Buffer should constitute two-thirds of the total volume at the DNA-binding step to support efficient binding of DNA to the DNA IQ™ Resin. During development of the Casework Extraction Buffer, this original recommendation was revisited, and experiments showed that there is no difference in isolation efficiency when following the original recommendation (two-thirds of the total volume) and the current recommendation (one-half of the total volume) with regards to the DNA IQ™ Lysis Buffer. Based on extensive experimentation, the recommendations were updated in the DNA IQ™ Casework Pro Kit for Maxwell® 16 Technical Manual #TM332 for both liquid samples and samples on solid supports to add 200µl of DNA IQ™ Lysis Buffer to each sample to maintain proper binding conditions throughout isolation.
Q: Should I expect any change in downstream performance of DNA isolated using the DNA IQ™ Casework Pro Kit for Maxwell® 16 and the Casework Extraction Buffer?
The preprocessing recommendations were changed to extract forensic samples, but the core DNA isolation chemistry has remained unchanged for more than a decade. The DNA IQ™ chemistry is well known for producing DNA characteristically low in inhibitors and impurities that might affect downstream amplification. During development of the Casework Extraction Kit, the purity and performance of DNA isolated from various sample types was assessed using the DNA IQ™ Casework Pro Kit for Maxwell® 16 through varied means. The Plexor® HY internal positive control (IPC) results were monitored to detect impurities that inhibit amplification. Samples were tested for residual alcohols from the isolation method. “Reagent blank” samples that did not contain a biological sample were extracted, then co-amplified with a known amount of a DNA sample in a quantitative PCR method (Plexor® HY System) or an endpoint PCR method (PowerPlex® 16 System). These results then were compared to results generated with the same DNA sample co-amplified with a volume of elution buffer (that was not processed using the DNA IQ™ Casework Pro for Maxwell® 16 cartridge). The results pointed to a clean isolation process optimized for use in downstream amplification.
Finally, a large panel of samples were extracted to show that if we could obtain a DNA concentration value from the Plexor® HY System, we could use this information to generate full profiles using an STR multiplex (PowerPlex® 16 HS System). In this way, the Casework Extraction Kit not only resulted in a higher DNA yield, as measured with the Plexor® HY System, but generated full STR profiles from a broad panel of sample types, representative of the sample types likely to be encountered in the field.