DNA Genotyping is a laboratory method for the identification of victims from a disaster or mass casualty events. The DNA Commission of the International Society for Forensic Genetics (ISFG) provided laboratory guidelines for sample collection, storage, DNA purification, and Genotyping. Software and bioinformatics tools that have been developed. Human Remains cannot be recovered from the field immediately and can be stored for variable periods of time and can be refrigerated or non-refrigerated.
Identification can be based on dental remains, anthropological evidence, and fingerprints. DNA is the most common identification. Samples must be shipped to offsite Forensic Labs. Decomposed human remains require highly sensitive methods. Nuclear DNA is highly preferred for Forensic Identification.
DNA Testing is used for human DNA Identification. It requires complex laboratory methods, instruments and also requires several months to years for analysis. The ANDE-Rapid DNA Identification system comprises ANDE-6C Instrument, A & I chips, Expert system software. The ANDE-6C Instrument received FBI NDIS approval for the processing of buccal swabs. This system utilized a multiplex assay that includes 27 STR loci (23 autosomal,3Y chromosomal, and Amelogenin loci).
NDIS approval is critical for the implementation of Rapid DNA Analysis instruments in the Police station. NDIS approval is critical for the implementation of the Rapid DNA Analysis machine in the police station. DNA IDs generated by ANDE can be used to search the FBI’s DNA database using the Rapid DNA Index System.
ANDE plays an important role in the identification of remains from the November 2018 campfire in California. Rapid DNA Index system was used to identify the majority of victims from hours to days following the recovery of dead bodies. Rapid DNA IDs were generated from unidentified remains and were searched against those generated from buccal swabs of family members.
Forensic Anthropology Center at the University of Tennesse is operating a research facility or body farm, an outdoor laboratory to study human decomposition. Individuals from around the world donate dead bodies to strengthen forensic science. Samples of hair, nails, and samples can be taken for ongoing research. Donors are placed at the Anthropology Research Facility and decomposition is monitored through photos and longitudinal sampling can occur through decomposition. The essay presents results on the Rapid DNA Identification on tissue samples.
Materials and Methods
Forensic Anthropology Center at the University of Tennesse provided deceased human subjects for testing and research using ANDE. Donors were 7 female and 3 male. Two of the ten donors who have been autopsied were used to provide the brain samples. Teeth were collected from 7 donors and were molars. Eight of the donors provided liver, buccal, muscle, bone samples. Liver samples (LD01 and LD08) were the liver samples. Buccal samples and blood cards were collected to use as a reference for STR genotyping. Anthropology Research Facility consists of 3 acres of land above the Tennesse river. Raccoons can be considered as a taphonomic factor (that alters the rate and pattern of decomposition). Donors were swabbed at different start and endpoint. The samples were sampled throughout the post-mortem period. Sampling continued for a year.
Sample types analyzed were as follows:
Buccal cells -Buccal swabs that contain 3 mg of buccal samples were taken. Some swabs included maggots inhabited the oral cavity aids n the decomposition.
Liver– 3 mg of wet tissue from the liver was taken from the right lobe of the liver for examination.
Brain– Brain tissue was deposited was on a piece of drywall to evaluate decomposition outside the body.
Skeletal Muscle-3 mg of red quadriceps and biceps tissues were obtained. Sample sites were covered with duct tape to eliminate instant access that affects the rate of decomposition.
Teeth-One premolar or molar teeth were separated and the root was separated for analysis.
Bone-Samples of approximately 500 mg of femur shaft and distant foot phalanges were the samples. Samples were immediately stored at -20C for analysis. Tissue swabs, Buccal swabs, Muscle tissues are thawed for 15 minutes prior to analysis in ANDE by insertion into either E or I chip.
Blood– A punch was taken and transferred into 2 ml microcentrifuge tubes. Punch macerated in TE buffer and allowing the material to get released into the substrate. Incubate at 50C for 15 minutes. ANDE swab inserted into the tube and analyzed on I chip.
Buccal lining– Samples were laid onto the Petri plate. Swabs or tissue fragments can be inserted into the swab chamber.
Muscle, Brain, and Liver– Fragments of muscles, liver, and brain tissues are fragmented and placed onto the swab chamber.
Bone– Drilled bone fragment is cleaned with water and lightly washed to remove dirt. The bone immersed in 10 % bleach solution and inverted 15-20 times. The bone fragment is then cleaned with ethanol and then inverted into 15-20 minutes. The bone is then crushed into bone powder and to this 120 µl of ANDE solution was added. The mix was vortexed for 1 minute. 15 µl is then used for Rapid DNA Identification. For older bones (100-500 mg) bone solution was added and incubated at 56C.
Tooth– Adhering tissues were cleaned and tooth fragments can be used for analysis.
Rapid DNA Processing- Rapid DNA Analysis includes automated instruments, Microfluidic chips, Expert Analysis system software, A and I chip. I chip can be used for identifying unidentified human remains and A chip for the Family Reference sample. Chips perform automated purification and amplification of 20 STR loci. Chemical reagents are added onto the chip and DNA ID is identified using Expert system software less than 2 hours. Chips are stable for 6 months.
Conventional Laboratory Testing
The results generated from conventional laboratory testing and the ANDE system were submitted to BODE Cellmark Forensics. Samples presented for testing include blood cards and fragments of skeletal muscles. All the samples were processed using Powerplex Fusion 6C and Powerplex 21.
Results and Discussion
Two types of microfluidic chip consumables utilized in the study- the ANDE A chip and I chip. ANDE A chip can process 5 samples in 94 minutes. I chips process up to 4 samples in 103 minutes.
Buccal swabs collected in Mass disaster setting aids in getting DNA ids. Failure to generate results is due to insect activity. Findings were confirmed by three dead bodies recovered from the mass disaster setting.
DNA ID’s obtained from the samples within 2-8 days with 5 of 6 samples generated partial or full DNA IDs. The number of loci decreased with increased exposure. On day 5 one of the samples generated 27 loci, on day 6 16 loci were available and no STR peaks observed by day 7. Some samples could not be collected due to a sample of tissue disintegration. All muscle samples generated full DNA IDs with refrigeration for the 3-month generation of morgue study.
DNA processing of swabbed liver generated folds of average signals due to variability in tissue disintegration.
Brain swabs or fragments collected from 2 donors and DNA IDs generated from all the samples on the 3rd day and DNA IDs generated from one of the samples on day 7.
Bone and Tooth
Bone fragments (83 of 87) generated full DNA IDs. Desiccated rib materials are difficult to process because they are fibrous for generating DNA IDs. More surface area was required to generate DNA IDs. Tooth processing was successful and all 24 samples generated DNA IDs.
Samples should be collected based on two criteria
Samples that are likely to collect and the samples that can be rapidly used to generate DNA IDs. Buccal swabs are easier to collect and Muscle swabs can be collected after 5 days of exposure to generate DNA IDs.
Rapid DNA Analysis processing system generates accurate DNA IDs in a fraction of time when compared to conventional processing. Buccal and tissue swabs generate IDs in less than 2 hours. Fresh bone generates IDs in 3 hours. Older bone sample results are generated overnight. Rapid DNA Analysis enables transportation time and reduces contamination issues from crime scene to lab. Rapid DNA analysis is a method used for the identification of human remains and results can be obtained in less than 2 hours. Non-technical users can also generate STR results.