Often in forensic casework, the DNA evidence collected can be degraded due to the environmental conditions it was collected from or the sample age. DNA extraction techniques over the past several years have been optimized to collect smaller DNA fragments from common forensic sample types such as fired cartridge casings, bone and rootless hair shafts. These types of forensic samples often contain very low quantities of DNA and are of poor quality, which can affect how well they are amplified with the current standard short tandem repeat (STR) megaplexes. These challenges can lead scientists to turn to other methods for degraded DNA like miniSTR analysis. However, these methods have some notable disadvantages regarding the statistical weight of the profiles generated or require consuming more DNA extract to amplify in multiple chemistries. Other non-STR technology for detection of short fragment DNA lack database comparisons for searching. The current 5 or 6-dye CE systems are not able to support as many or as small an average amplicon size as would be required to generate profiles with greater completeness and discriminatory power from degraded DNA. By leveraging the extended 8-dye chemistry, the Spectrum CE system can address common limitations of typing degraded DNA by enabling the inclusion of more miniSTRs without compromising the discriminatory power of STR megaplex assays.
In this study, Promega’s PowerPlex 35GY (35GY) amplification kit compatible with the Spectrum CE 8-dye system was evaluated and compared with Promega’s PowerPlex Fusion 6C (Fusion 6C) amplification kit to determine if more genetic information could be obtained from degraded forensic samples. The 35GY system is a multiplex amplification assay that uses the expanded 8-dye chemistry to target 35 loci in a single assay. The 35GY kit contains all loci found in the Fusion 6C kit but redesigned to decrease the amplicon size of the largest autosomal loci.
Bones, handled fired cartridge casings, and rootless hair shafts were collected to be analyzed as mock evidence to mimic often degraded casework samples. In addition, two artificially degraded sample sets were tested, one using UV radiation and the other using the Covaris M220 Focused-ultrasonicator, to further evaluate the detection capabilities of 35GY with degraded sample material. A sensitivity study testing a range of input quantities as well as an inhibition study testing the effects of common inhibitors were included to further evaluate the detection capability of 35GY. All extracts were amplified in both 35GY and Fusion 6C and analyzed on the Spectrum CE and 3500xl Genetic Analyzer, respectively.
All profiles generated with 35GY showed significant improvement compared to the traditional 6-dye chemistry, supporting the theory that the redesigned primers for smaller amplification fragment length can improve profile results from degraded DNA. With the overall increase in profile completeness, the match statistics also increased favorably for those partial profiles with 35GY.
Overall, the expanded 8-dye chemistry provides a promising solution to overcoming the common challenges of typing degraded forensic samples in a workflow that maintains the current industry standard of STR amplification and CE analysis.
Often in forensic casework, the DNA evidence collected can be degraded due to the environmental conditions it was collected from or the sample age. DNA extraction techniques over the past several years have been optimized to collect smaller DNA fragments from common forensic sample types such as fired cartridge casings, bone and rootless hair shafts. These types of forensic samples often contain very low quantities of DNA and are of poor quality, which can affect how well they are amplified with the current standard short tandem repeat (STR) megaplexes. These challenges can lead scientists to turn to other methods for degraded DNA like miniSTR analysis. However, these methods have some notable disadvantages regarding the statistical weight of the profiles generated or require consuming more DNA extract to amplify in multiple chemistries. Other non-STR technology for detection of short fragment DNA lack database comparisons for searching. The current 5 or 6-dye CE systems are not able to support as many or as small an average amplicon size as would be required to generate profiles with greater completeness and discriminatory power from degraded DNA. By leveraging the extended 8-dye chemistry, the Spectrum CE system can address common limitations of typing degraded DNA by enabling the inclusion of more miniSTRs without compromising the discriminatory power of STR megaplex assays.
In this study, Promega’s PowerPlex 35GY (35GY) amplification kit compatible with the Spectrum CE 8-dye system was evaluated and compared with Promega’s PowerPlex Fusion 6C (Fusion 6C) amplification kit to determine if more genetic information could be obtained from degraded forensic samples. The 35GY system is a multiplex amplification assay that uses the expanded 8-dye chemistry to target 35 loci in a single assay. The 35GY kit contains all loci found in the Fusion 6C kit but redesigned to decrease the amplicon size of the largest autosomal loci.
Bones, handled fired cartridge casings, and rootless hair shafts were collected to be analyzed as mock evidence to mimic often degraded casework samples. In addition, two artificially degraded sample sets were tested, one using UV radiation and the other using the Covaris M220 Focused-ultrasonicator, to further evaluate the detection capabilities of 35GY with degraded sample material. A sensitivity study testing a range of input quantities as well as an inhibition study testing the effects of common inhibitors were included to further evaluate the detection capability of 35GY. All extracts were amplified in both 35GY and Fusion 6C and analyzed on the Spectrum CE and 3500xl Genetic Analyzer, respectively.
All profiles generated with 35GY showed significant improvement compared to the traditional 6-dye chemistry, supporting the theory that the redesigned primers for smaller amplification fragment length can improve profile results from degraded DNA. With the overall increase in profile completeness, the match statistics also increased favorably for those partial profiles with 35GY.
Overall, the expanded 8-dye chemistry provides a promising solution to overcoming the common challenges of typing degraded forensic samples in a workflow that maintains the current industry standard of STR amplification and CE analysis.
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Validation and Implementation Manager, Bode Technology
Heather Sarik is the Validation and Implementation Manager at Bode Technology. Heather has been a part of the Bode team since 2012 where her love for learning and troubleshooting led her to join the validations team in 2014. She enjoys being involved in all aspects of laboratory processing and specializing in automation workflows.
