Post mortem modifications in DNA and estimation of PMI.pptx

Post mortem modification of human dna and its significance on estimation of PMI

1. Post Mortem Modification of Human DNA and its significance on estimation of Post-Mortem Interval Dr. Satrajit Roy Junior Resident (3 rd year) Upgraded Department of FSM Medical College, Kolkata Prof. (Dr.) Chandan Bandyopadhyay Professor and Head of the Department Upgraded Department of Forensic and State Medicine Medical College, Kolkata

2. OBJECTIVES

3. CONTENTS Introduction Biochemical changes in DNA after death Factors influencing post-mortem DNA degradation Current techniques, if any, for analyzing the stability and rate of degradation of DNA and the feasibility of such procedures Correlation between DNA degradation and time since death Reliability, limitations and admissibility before court of law

4. INTRODUCTION Estimating the time since death requires the calculation of measurable data along a time-dependent curve back to the starting point The majority can be divided into two main categories: those occurring within the early post-mortem period and those during the late post-mortem period

5. Biochemical changes in DNA after death 1. Immediate Postmortem Changes (0–30 minutes after death) Cessation of DNA Repair Mechanisms: Hypoxia and pH Drop: 2. Early Postmortem Phase (Hours to Days) A. Autolysis (nuclease activation and protease activity) B. Hydrolytic damage (depurination/deamination/oxidative damage)

6. Cont. 3. Intermediate Phase (Days to Weeks) A. Microbial and Putrefaction Effects (proliferation & action of saprophytic org) B. Chemical Modifications (crosslinking and OXIDATIVE DAMAGE) 4. Late Postmortem Phase (Months to Years) A. Environmental Factors (temperature/humidity/radiation) B. Long-Term Degradation Pathways (fragmentation/diagenesis/mineralization)

7. Factors influencing post-mortem DNA degradation Factor Effect on DNA Degradation Temperature Higher temps → faster decay (Q₁₀ rule: ~2x rate per 10°C rise). pH Acidic conditions → depurination; alkaline → strand breaks. Oxygen Aeration → oxidative damage; anoxia → slower decay. Water Activity Moisture → hydrolysis; desiccation → slows enzymes. Microbial Load High microbial activity → rapid DNA breakdown. Tissue Type Bones/teeth preserve DNA longer than soft tissues.

8. Venturing into the field of forensic science To narrow the time frame to minutes, if possible, or to hours (creating a precise “molecular clock”) An objective measure that occurs regardless of external influences In scenarios where application of traditional methods are limited or have minimal/no significance Identification Ancient DNA studies Degradation studies

9. Post-mortem stability of DNA? Whether degradation occurs at a particular rate? Can this rate be determined using laboratory methods? Specificity and Sensitivity of the procedures employed and the test results? Feasibility of such procedures in underdeveloped/developing countries? Questions that needs to answered…

10. Stability and degradation of DNA in the post-mortem period Degradation of DNA begins directly after death, when the body’s natural DNA repair mechanisms are halted (Parsons and Weedn , 1996) There are three main ways in which the DNA molecule degrades in a living person: hydrolysis, oxidation, and methylation.

11. Aspects DNA degradation in living organisms DNA degradation in dead organisms Mechanisms 1) Nucleases involved in DNA replication, repair, and apoptosis 2) Autophagy 3) Oxidative stress 4) DNA repair pathways 1) Autolysis and post-mortem processes 2) Nucleases released from the lysed cells 3) Microbial enzymes 4) Environmental factors (temperature, humidity, ultraviolet radiation) DNA  fragmentation Possible DNA fragmentation due to enzymatic cleavage DNA fragmentation often accompanies DNA degradation Ce llular regulation Tightly regulated and controlled to maintain genomic integrity No active cellular regulation or control Importance Ensures proper DNA replication, repair, and programmed cell death Implications for forensic science and paleogenomics Differences between DNA degradation in living and dead organisms

12. POLYMERASE CHAIN REACTION

13. Techniques for analysis of post-mortem changes in DNA behaviour 1. Gel electrophoresis for fragment size. 2. qPCR for quantifying DNA and amplifying fragment length.(dec/ inc ) 3. Next-gen sequencing to assess fragment distribution. 4. Chemical assays (HPLC, Mass Spec) for base modifications. 5. FTIR for structural changes. 6. DNA hybridization efficiency. (dec/ inc ) 7 . TUNEL assay for PM fragmentation. 8 . Mitochondrial vs nuclear DNA ratios. 9 . Telomere analysis (maybe). 10. Microbial community analysis. 11. Mathematical modeling of degradation rates.

14. Degradation study Over the years, a number of degradation studies have been performed with varying success with development of better molecular techniques. In 1988, Perry et al. examined DNA degradation in human rib bone with the goal of establishing PMI. It appeared that the variation between samples of different individuals was less than variation caused by natural factors such as humidity.

15. Stability and rate of DNA degradation with time According to Bär et al. (1988:68-69), the best stability after the longest period (3 weeks) occurs in the “brain cortex, lymph nodes, and psoas muscle.” Using minisatellites, it was found that DNA retained its structure in the spleen and kidney for five days before badly degrading. The blood results were inconsistent ; clots yielded large amounts of DNA

16. At the time of the Johnson and Ferris (2002) study, the best approximation of PMI only came within an eight-hour interval. They used single-cell gel electrophoresis, or the comet assay, to reveal a definitive DNA degradation pattern that is “organ and time-dependent” (Johnson and Ferris, 2002:46).

17. Correlation between rate of DNA degradation and estimation of PMI CHALLENGE??

18. IT IS NOT THAT EASY!!! Mechanisms of DNA degradation after death Environmental and contextual variables Tissue-specific degradation rates Key metrics to assess the quality and quantity of degraded DNA Statistical and computational models Other challenges and limitations Epigenetic clock Microbiome succession

19. Reliability, limitations and admissibility Environmental and climatic factors Technological and resource availability Baseline data and research Training and expertise Validation, acceptance and chain of custody Comparative effectiveness with RNA degradation Logistics

20. Notable research works Anderson et al. (2005): evaluated the degradation of nuclear and mitochondrial DNA in human muscle and blood tissues kept under “controlled environmental conditions” Sampietro et al. (2006): analyzed DNA degradation (from bones dated 200-2000 years) in ancient samples to estimate time since death. Damato et al. (2012): assessed the rate of DNA degradation in blood stains Schmedes et al. (2020): focused on DNA degradation in soft tissues over time in varying climates.

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23. ACKNOWLEDGEMENT

24. Thank You….