Applications in forensics PCR and DNA fingerprinting.pptx

Applications in forensics PCR and DNA fingerprinting.pptx

1. 30/11/23 Applications in forensics: PCR and DNA fingerprinting Dr. U. Venkateswara Prasad MSc., PhD. Assistant Professor School of Life Science B.S.Abdur Rahman University, Chennai-600048

2. INTRODUCTION TO FORENSIC SCIENCE ROLE OF DNA FINGERPRINTING AND PCR INTRODUCTION TO DNA FINGERPRINTING STEPS OF DNA FINGERPRINTING ADVANTAGES INTRODUCTION TO PCR STEPS OF PCR ADVANTAGES OF PCR 2

3. What is Forensic Science? Forensic science (or forensics) is the application of scientific knowledge to answer questions of interest within the legal system. Evidence from accidents and crime scenes is collected and analyzed by forensic scientists. Forensic scientists act as expert witnesses.

4. Forensic Scientists Use Techniques from Many Scientific Disciplines Southern Blotting Polymerase Chain Reaction (PCR) Blood Typing Anthropology – Analyze remains in advanced stages of decomposition Genetics/Molecular Biology – Uses biological material to determine serological and DNA profiles Toxicology – Identifies poisons, drugs and/or alcohol in tissues and body fluids. Chemistry – Analysis of trace physical evidence Pathology – Determine cause of death post-mortem

5. Forensic Science Lessons Engage Critical Thinking and Literacy Skills Read witness statements and police report Analyze evidence using forensic science techniques Write a persuasive essay using the evidence from the written documents and the biological and physical tests.

6. DNA Sequences are Unique The chances of two individuals having exactly the same DNA profile is ~30,000 million to one Single Nucleotide Polymorphisms (SNPs) Restriction Fragment Length Polymorphisms (RFLPs) DNA profiling is a forensic technique used to identify individuals by analyzing differences within DNA. CODIS – Combined DNA Index System

7. A technique used by scientists to distinguish between individuals of the same species using only samples of their DNA. DNA fragments show unique patterns from one person to the next. Used in paternity disputes and as forensic evidence. Historic Examples: 1800 Photographs Early 1900s: fingerprints, Palm prints retinal scan Foot wear and tire imprssions , Ear, lips etc. DNA Fingerprinting 1985: United Kingdom The process of DNA fingerprinting was invented by-Sir Alec Jeffreys , Dr. peter Gill Dr. Dave Werrett at the University of Leicester in 1985.

8. DNA is Isolated from Crime Scene Samples Cell membranes are disrupted to release its intracellular components. The resulting “lysate” consists of cytoplasm, metabolites, DNA, RNA, proteins and organelles. Want to learn more about forensic blood testing?

9. Chemical Structure of DNA is always the same. Important: The order of the base pairs is different! 3 Billion Nucleotides form our 46 chromosomes. In fact, no two people on Earth have exactly the same genetic sequence, except identical twins. Important: Every cell contains a copy of our DNA!

10. VNTR(variable number of tandem repeats)- recognition that variable numbers of repeated nucleotides can be found in DNA and can be used for identification of individuals. VNTR’s provide a scientific marker for identity. How does it Work? DNA Fingerprinting using VNTR's Cut out the segment of the chromosome containing this variable number of tandem repeats ( VNTR's ). Identify the VNTR's for the DNA sequence of the repeat.

11. Isolation of DNA DNA must be recovered from the cells or tissues of the body. Blood Hair Saliva Semen DNA samples have been obtained from vaginal cells transferred to the outside of a condom during sexual intercourse. Only a small amount of tissue is needed. For example, the amount of DNA found in one drop of blood or in one hair root is enough.

12. Stages of DNA Fingerprinting Step 1: Cells are broken down to release DNA. If only a small amount of DNA is available it can be amplified using the polymerase chain reaction (PCR).

13. RFLP – Restriction Fragment Length Polymorphism More accurate than PCR Requires a large sample of fresh DNA Takes longer time & costly than PCR When a match if found, there is no question as to whether the suspect was at the scene. PCR – Polymerase Chain Reaction Takes less time, less costly Requires only a small sample That sample is amplified using polymerases to force the small amounts of DNA to copy themselves many times. Can be performed on older samples If no match is found, the donor was not at the scenes

14. PCR Amplifies Specific DNA Sequences Denaturation (94°C) Double-stranded DNA is “unzipped” into single strands. Annealing (40-65°C) Primers base pair with the target DNA sequence. Extension (72°C) Taq polymerase extends the primer and synthesizes a new strand of DNA.

15. RFLPs Are Used as Landmarks for DNA Fingerprinting Restriction Fragment Length Polymorphisms (or RFLPs) are heritable differences in the nucleotide sequence. Some RFLPS add a restriction enzyme cut site to the DNA.

16. Electrophoresis DNA fragments are injected into wells and an electric current is applied along the gel. DNA is negatively charged so it is attracted to the positive end of the gel. The shorter DNA fragments move faster than the longer fragments. DNA is separated on basis of size.

17. Step 2: Cutting, Sizing, and Sorting Special restriction enzymes are used to cut the DNA at specific places. The DNA pieces are sorted according to size by a technique called electrophoresis. The DNA pieces are passed through a gel made from seaweed agarose.

18. RFLPs Are Used as Landmarks for DNA Fingerprinting Variable Number of Tandem Repeats (or VNTRs) comprise short, repetitive DNA sequences present in multiple copies between two restriction sites 15-35 base pair sequences VNTRs appear between five and 100 times. Cat. # 334

19. DNA Fingerprinting is Used to Solve Crimes Forensic scientists identified a biological sample at the scene of a crime. DNA was extracted from the sample, amplified by PCR and digested with restriction endonucleases to create a DNA Fingerprint. A match suggests that the suspect was at the crime scene. Cat. # 109 Cat. #130

20. 1. Paternity Test By comparing the DNA profile of a mother and her child it is possible to identify DNA fragments in the child which are absent from the mother and must therefore have been inherited from the biological father. Application’s

21. 2. DNA Profiling can solve crimes The pattern of the DNA profile is then compared with those of the victim and the suspect. If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene. If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.

22. PCR Reaction requirements Template DNA – total genomic DNA isolated from an organism that contains a target region to be amplified DNA primers - Short pieces of single-stranded DNA that flank the target Taq DNA polymerase - Attaches nucleotides on the growing strand of DNA Nucleotides (GATC) – Polymerase adds complementary nucleotides to the template

23. PCR Reactions are placed in a machine called a thermal cycler. The machine cycles through three temperatures .

24. PCR Heat samples to 94°C for a minute or so to denature the double stranded template DNA.

25. PCR The thermal cycler repeats the denaturing, annealing, and elongating temperatures approximately 30 times.

26. PCR PCR amplification is logarithmic, meaning the number of copies of the target is doubled every cycle.

27. Famous Cases Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence. He was arrested in 1986 for the rape and murder of two girls. O.J. Simpson was cleared of a double murder charge in 1994 which relied heavily on DNA evidence. This case highlighted lab difficulties. In 2002 Elizabeth Hurley used DNA profiling to prove that Steve Bing was the father of her child Damien.

28. 3. Personal Identification The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed. Diagnose inherited disorders in both prenatal and new born babies. By studying the DNA fingerprints of relatives who have a history of some particular disorder. Identify DNA patterns associated with the disease. These disorders may include hemophilia, Alzheimer's, sickle cell anemia, thalassemia, and many others. 4.Diagnosis of Inherited Disorders

29. Drawbacks Human Error Sneezes, improper storage, failure to label samples Chain of Custody Collection of evidence must be systematically recorded and access to evidence must be controlled. Collecting samples High temperatures can degrade DNA Bacteria Moisture

30. References Russel, P.J. 1998 Genetics (5 th edition). Chawla, H.S.2005 Introduction to Plant Biotechnology(2 nd edition) Singh, B.D. 2005 Biotechnology Expanding Horizons http://www.biologycorner.com/bio4/notes/DNA_fingerprint. http://www.scq.ubc.ca/a-brief-tour-of-dna-fngerprinting/ http://evolution.berkeley.edu/evolibrary/news/060301_crime

31. Thank you Mail id: [email protected]