Abstract Title

Effect of Detergent Selection on Quantity of DNA Extracted and on STR Profile Developed from Bone-Derived DNA

Presenter Name

F. Nicole Proctor

RAD Assignment Number

1302

Abstract

DNA analysis is often essential to make positive associations in cases of unidentified persons, missing persons, and mass fatality incidents. In cases such as these, human skeletal remains are frequently the only source of genetic material available, but bone-derived DNA characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types.

Conventional DNA extraction methods were developed based on the biochemical composition of soft tissues or body fluids. DNA from these unmineralized sample types is more readily extracted and contains fewer inhibitors than DNA found in bone; furthermore, skeletal remains encountered in casework may be aged or subjected to environmental factors that reduce the quality of DNA obtained. These sample-specific issues support the development of specialized extraction techniques for bone in order to obtain the highest quantities of DNA and improved quality STR profiles.

Prior work has demonstrated that increased DNA quantities are obtained from human skeletal remains when using Buffer ATL in conjunction with Collagenase Type II (CLSII) enzyme. It has also been determined that metals, particularly calcium, copurify with DNA when processing bone samples. These copurified metals have been shown to inhibit PCR amplification of STR markers. Building upon these findings, a protocol was designed to determine whether use of a detergent other than Buffer ATL would continue to improve upon current methods for DNA extraction from human bone.

An unembalmed human cadaver diaphysis was obtained through the Willed Body Program of the University of North Texas Health Science Center. DNA was purified on the EZ1® Advanced XL System (Qiagen®, Hilden, Germany) after employing the modified digestion step. The DNA isolates were quantified using the Investigator® Quantiplex HYres Kit (Qiagen®), then STR markers were amplified using the Investigator® 24Plex QS Kit (Qiagen®) and fragment analysis performed with the 3500xL Genetic Analyzer (Thermo Fisher Scientific, Inc., Carlsbad, CA). STR profiles were assessed using GeneMapper® ID-X v1.4 (Thermo Fisher Scientific). All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA) and RStudio® (RStudio® Inc., Boston, MA).

Though Buffer ATL yielded significantly higher quantities of DNA per milligram of bone, results indicate that using an increased strength anionic detergent, such as SDS or SLS, in conjunction with CLSII enzyme will improve the quality of STR profiles produced from human skeletal remains. Full profiles were recovered for all concentrations of SDS and SLS, while allelic drop out was observed for Buffer ATL and Triton™ X-100. Mean peak heights of profiles produced using all concentrations of SDS and SLS represented a quantitative improvement over both Buffer ATL and Triton™ X-100. Mean peak height ratios of profiles produced using all concentrations of SDS and SLS also represented a qualitative improvement compared to samples digested using CLSII enzyme with Buffer ATL or Triton™ X-100. Unlike SDS, there are no special considerations for storage or handling of SLS detergent solutions, making it an excellent choice for use in forensic laboratories. Use of SLS consistently produces sufficient quantities of DNA and full STR profiles at all concentrations tested, and as no significant differences were observed between concentrations of SLS, the lowest tested concentration of 1% should be employed in order to conserve resources.

Presentation Type

Poster

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Effect of Detergent Selection on Quantity of DNA Extracted and on STR Profile Developed from Bone-Derived DNA

DNA analysis is often essential to make positive associations in cases of unidentified persons, missing persons, and mass fatality incidents. In cases such as these, human skeletal remains are frequently the only source of genetic material available, but bone-derived DNA characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types.

Conventional DNA extraction methods were developed based on the biochemical composition of soft tissues or body fluids. DNA from these unmineralized sample types is more readily extracted and contains fewer inhibitors than DNA found in bone; furthermore, skeletal remains encountered in casework may be aged or subjected to environmental factors that reduce the quality of DNA obtained. These sample-specific issues support the development of specialized extraction techniques for bone in order to obtain the highest quantities of DNA and improved quality STR profiles.

Prior work has demonstrated that increased DNA quantities are obtained from human skeletal remains when using Buffer ATL in conjunction with Collagenase Type II (CLSII) enzyme. It has also been determined that metals, particularly calcium, copurify with DNA when processing bone samples. These copurified metals have been shown to inhibit PCR amplification of STR markers. Building upon these findings, a protocol was designed to determine whether use of a detergent other than Buffer ATL would continue to improve upon current methods for DNA extraction from human bone.

An unembalmed human cadaver diaphysis was obtained through the Willed Body Program of the University of North Texas Health Science Center. DNA was purified on the EZ1® Advanced XL System (Qiagen®, Hilden, Germany) after employing the modified digestion step. The DNA isolates were quantified using the Investigator® Quantiplex HYres Kit (Qiagen®), then STR markers were amplified using the Investigator® 24Plex QS Kit (Qiagen®) and fragment analysis performed with the 3500xL Genetic Analyzer (Thermo Fisher Scientific, Inc., Carlsbad, CA). STR profiles were assessed using GeneMapper® ID-X v1.4 (Thermo Fisher Scientific). All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA) and RStudio® (RStudio® Inc., Boston, MA).

Though Buffer ATL yielded significantly higher quantities of DNA per milligram of bone, results indicate that using an increased strength anionic detergent, such as SDS or SLS, in conjunction with CLSII enzyme will improve the quality of STR profiles produced from human skeletal remains. Full profiles were recovered for all concentrations of SDS and SLS, while allelic drop out was observed for Buffer ATL and Triton™ X-100. Mean peak heights of profiles produced using all concentrations of SDS and SLS represented a quantitative improvement over both Buffer ATL and Triton™ X-100. Mean peak height ratios of profiles produced using all concentrations of SDS and SLS also represented a qualitative improvement compared to samples digested using CLSII enzyme with Buffer ATL or Triton™ X-100. Unlike SDS, there are no special considerations for storage or handling of SLS detergent solutions, making it an excellent choice for use in forensic laboratories. Use of SLS consistently produces sufficient quantities of DNA and full STR profiles at all concentrations tested, and as no significant differences were observed between concentrations of SLS, the lowest tested concentration of 1% should be employed in order to conserve resources.