Rapid Processing of Casework Samples Using the Casework Direct Kit, Custom
Written by Erica K. Graham, Mary Loten, Jonelle M. Thompson, Jon Drobac, & Anupama Gopalakrishnan, Promega Corp   

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The forensic casework DNA typing workflow consists of DNA extraction, DNA quantification, PCR amplification, detection of PCR products and analysis (1). Forensic samples typically require separation of the DNA from a physical substrate, such as a swab, in addition to DNA extraction—cell lysis and release of DNA from the nucleus. Conventional DNA extraction methods purify DNA by isolating it from other cellular components and potential PCR inhibitors. The goal of extraction is to produce high-quality DNA of sufficient yield for use in downstream DNA typing applications (1). One challenge to producing an optimal DNA extract is obtaining adequate human DNA yield and concentration from casework samples that normally contain minimal amounts of DNA.

The Casework Direct Kit, Custom (Cat.# AX4560), contains a buffer and a reducing agent designed to rapidly produce a DNA lysate from forensic casework samples of varying substrates and size (2). The Casework Direct Kit, Custom protocol generates amplification-ready lysates in approximately 35 minutes and is compatible with many PowerPlex® amplification systems. No purification is required before using the lysate with downstream STR amplification unless inhibition is indicated by the quantification data. Since the method does not involve any binding or washing, there is less potential for loss of DNA during the process. This method is particularly useful for touch samples and sexual assault-type evidence samples containing low quantities of male DNA. The protocol also reduces the amount of hands-on time for the DNA analyst, thereby reducing contamination risk.

The Casework Direct lysate can be evaluated with PowerQuant® or Plexor® HY Systems to quantify the abundance of human DNA, determine the male/female DNA ratio and predict PCR inhibition (2). Additional information regarding the quality of DNA is obtained when the lysate is evaluated using the PowerQuant® System. Informed decisions about the next step in the workflow can then be made about each Casework Direct sample.

Protocol for Casework Direct Kit, Custom
The protocol is simple and straightforward, with a brief incubation step followed by centrifugation. The lysate is ready for use in the quantification assay and STR amplification.

Figure 1. Protocol for Casework Direct Kit, Custom.

Y-Screening Application
The Casework Direct Kit, Custom, is ideal for screening sexual assault-type evidence samples for the presence of male DNA, known as Y-screening. Traditional serology screening tests are less sensitive than DNA typing kits and are time consuming (3). Reliance on the results from serology tests could miss potential CODIS-eligible DNA profiles (3). A recent escalation in legislation requiring mandatory testing of all sexual assault evidence kits has resulted in an increase in laboratory backlogs and turnaround time (4). Use of the Casework Direct Kit, Custom, in conjunction with a DNA quantification system allows the laboratory to make prompt decisions about which STR amplification system is most appropriate and can provide justification for ceasing work on a sample when no male DNA is present.

In the experimental data presented in Table 1, a variety of mock casework samples containing semen and prepared on various substrates were processed (5). The samples were tested using serology screening methods, Casework Direct Y-screening and the PowerPlex® Fusion 6C and PowerPlex® Y23 Systems. All samples tested positive for prostate-specific antigen (PSA) and male DNA via Y-screening with the Casework Direct Kit, Custom. Full Y-STR profiles and partial or full autosomal STR profiles were obtained from all samples except where inhibition is stated. The blood/semen mixture on black nylon and the blood/semen mixture on red nylon/polyester generated STR profiles that were inhibited and exhibited a noisy baseline. Both of these samples exhibited internal PCR control (IPC) Cq shift flags with the PowerQuant® System. The inconclusive acid phosphatase (AP) results were due to substrate color that masked the reagent color change. Substrates containing heavy soil, such as fingerprint powder, may necessitate longer spin times in the basket.

Table 1. Serology, PowerQuant® System quantification and STR percent profile results for a variety of mock casework samples containing semen. Orange cells represent a threshold flag with the PowerQuant® System. Inc = inconclusive.

Touch DNA Application
Over the last few years, law enforcement agencies have seen an uptick in property crime evidence submissions, resulting in the need to use significant laboratory resources to test evidence for “touch DNA”—microscopic skin cells containing DNA that naturally rub off when an object, like a car steering wheel, is touched. When DNA is found at a property crime scene, laboratories are often able to match it to a known offender’s sample in CODIS.

In the experiment shown in Figures 2 and 3, ten touch DNA samples were processed with the Casework Direct Kit, Custom and Maxwell® FSC DNA IQ™ Casework Kit on the Maxwell® FSC Instrument (5). Total human DNA yields, as well as autosomal DNA concentrations, were compared. The Casework Direct Kit yielded more total human DNA than the Maxwell® FSC DNA IQ™ Casework Kit for nine out of the ten samples. The average concentration of autosomal DNA detected with the PowerQuant® System was similar between the two extraction methods with the exception of the telephone swab, which yielded more DNA with the Maxwell® FSC DNA IQ™ Casework Kit than with the Casework Direct Kit. No IPC Cq shift flag was detected in any of the samples.

Figure 2. Average total human DNA yield obtained for ten touch DNA samples extracted with both Casework Direct Kit, Custom, and Maxwell® FSC DNA IQ™ Casework Kit. The X axis represents the sample type. The Y axis represents average total DNA yield. The error bars represent +/– 1 standard deviation. Samples were quantified with PowerQuant® System on the Applied Biosystems® 7500 Real-Time PCR System.

Figure 3. Average autosomal quantification results for ten touch DNA samples extracted with both Casework Direct Kit, Custom, and Maxwell® FSC DNA IQ™ Casework Kit. The X axis represents the sample type. The Y axis represents average autosomal DNA concentration. The error bars represent +/– 1 standard deviation. Samples were quantified with PowerQuant® System on the Applied Biosystems® 7500 Real-Time PCR System.

Conclusion
The Casework Direct Kit, Custom, was developed for the rapid and efficient extraction of low-template DNA forensic casework samples. The protocol minimizes potential loss of DNA from samples and reduces the amount of time a DNA analyst handles a sample. The Casework Direct lysates can be successfully coupled with downstream quantification and PCR amplification using Promega systems.

The Casework Direct Kit, Custom, protocol in conjunction with the PowerQuant® System offers a sensitive, quick and informative Y-screening method for male DNA. The Casework Direct screening method is also suitable with touch DNA samples for use in deciding subsequent analysis steps in a forensic laboratory workflow.

To learn more about the Casework Direct Kit, Custom, please visit: www.promega.com/CaseworkDirect

References
1. Butler, J.M. (2012) Advanced Topics in Forensic DNA Typing: Methodology. Academic Press, Elsevier.
2. Rapid Processing of Swabs from Casework Samples Using Casework Direct Kit, Custom. Application Note #AN300, Promega Corporation.
3. Scientific Working Group on DNA Analysis Methods (December 2016) Recommendations for the efficient DNA processing of sexual assault evidence kits.
4. Gordon Thomas Honeywell Governmental Affairs (August 2016) August 2016 Updates
5. Graham, Erica K., et al. Developmental Validation of the Casework Direct Kit Custom: A Method for the Rapid Processing of Casework Samples. White Paper #WP108, Promega Corporation.

 
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