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Chapter 13 Forensic DNA

Chapter 13 Forensic DNA. Students will understand DNA’s role in Forensics science Students will know the different procedures for DNA profiling. I. DNA: A Forensic Science Tool. A. General Concepts 1. DNA is D eoxyribo n ucleic A cid

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Chapter 13 Forensic DNA

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  1. Chapter 13Forensic DNA Students will understand DNA’s role in Forensics science Students will know the different procedures for DNA profiling

  2. I. DNA: A Forensic Science Tool • A. General Concepts • 1. DNA is Deoxyribonucleic Acid • 2. The human genome project determined portions of the DNA structure of certain genes may be as unique as fingerprints. • Discovered by Alec Jeffreys et. al. at Leicester University, England • Called it DNA fingerprinting • Also called DNA profiling and DNA typing

  3. I. DNA: A Forensic Science Tool • B. What is DNA • 1. Chromosomes • 2. Almost 100,000 genes per chromosome • 3. Genes are the fundamental unit of heredity • 4. DNA construction • Polymer, a large molecule made by linking together a series of repeating units. • Nucleotides are the linked molecules with one phosphate, one sugar, and one nitrogen base. • It has a double helix shape with A bonded to T and C bonded to G

  4. I. DNA: A Forensic Science Tool • C. DNA at work • 1. DNA is identical due to Replication • 2. Inherited traits are viewed by the production of specific proteins • 3. Proteins are made up of a combination of 20 amino acids • 4. Amino Acid sequences aid in determining the genetic code • Codons (3 base pairs) match with tRNA anticodons which are connected to a single amino acid • This codon can be traced back to the DNA codes for the entire protein

  5. I. DNA: A Forensic Science Tool • 5. Single base changes can change the entire protein ex. Hemoglobin and sickle cell (glutamate to valine)

  6. I. DNA: A Forensic Science Tool • D. DNA Replication • 1. DNA unwinds • 2. New nucleotides attach to the “free” nucleotides • 3. Once the entire strand is unwound and all of the “free” nucleotides are matched, there are two exact DNA copies • 4. Scientists can copy DNA outside of the cell using DNA Polymerases • Called PCR (Polymerase Chain Reaction) • Accomplished in a DNA Thermal Cycler • each cycle doubles the DNA • After 30 cycles there can be over a billion strands

  7. DNA Polymerase: Unzipping DNA

  8. DNA Polymerase: Unzipping DNA

  9. I. DNA: A Forensic Science Tool • E. Recombinant DNA • 1. Cutting and splicing sections of DNA • 2. This can take advantage of known sections of DNA • Know the specific site of production • 3. Recombinant DNA relics on certain chemicals to cut DNA (called restriction enzymes) • The fragments can be incorporated into another DNA strand • There are 150 commercially used restriction enzymes • 4. Insert the DNA fragment into a bacterium strand with a different restriction enzyme (called splicing) • 5. Alters the bacteria’s DNA

  10. Recombinant DNA

  11. Recombinant DNA

  12. Electro-Gel

  13. Electrophoresis

  14. Electrophoresis

  15. I. DNA: A Forensic Science Tool • F. DNA Typing • 1. Restriction Fragment Length Polymorphism (RFLP) • a. Not all DNA segments code for proteins • b. Some segments occur as repeated letter sequences • Called “Tandem repeats” • c. Forensics use tandem repeats to identify subjects • Called DNA Typing • d. The number of repeating sequences within each pair may be different • Demonstrating differences between the strands from both parents

  16. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • d. (cont) • Also in a population the number of repeating strands are numerous • Increases with greater number of chromosomes • e. Restriction enzymes can be used to cut chromosomes into different fragments with different lengths • Are called restriction fragment length polymorphisms (RFLPs) • Long in length • Fragments are sorted by electrophoresis • Small fragments move faster than large fragments • The double stranded fragments are treated for separation • Fragments are transferred to Nylon membrane called Southern Blotting

  17. RFLP process from start to finish

  18. RFLP

  19. Restriction Fragment Length Polymorphism (RFLP)

  20. RFLP: Determine the relationships

  21. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • e. (cont) • Labeled with radioactive base pairs to identify complimentary strands (hybridization) • X-rayed to find pairs • When comparing look for matching bands • First scientifically accepted protocol in the U.S.

  22. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • 2. Polymerase Chain Reaction (PCR) • A second technique • More viable • Increased sensitivity • As little as 1 billionth of a gram of DNA • 50 times less • DNA polymerase can synthesize a specific region of DNA

  23. PCR

  24. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • 2. Polymerase Chain Reaction (PCR) • A DNA primer is used to prep the segment • Heat DNA strand (94°C) • DNA separates • Add specific sequences (primer) to the separate strands • Combine to the strands, while lowering the temp. • Add DNA polymerase and some free nucleotides • Reheat the tube (72°C) • Polymerase directs the rebuilding of double stranded DNA • Extends the primers

  25. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • 2. Polymerase Chain Reaction (PCR) [cont] • Doubles the number of DNA strands • Continue doubling process until significant number of copies are present • Makes genetic typing easier • First use was with the HLA DQ alpha system, (now DQA1) • Has significant numbers of variants • The process is easier than RFLP • DNA is extracted • Primer, DNA polymerase and free nucleotides are added

  26. PCR

  27. PCR

  28. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • 2. Polymerase Chain Reaction (PCR) [cont] • Heated and cooled • Select DNA is added to Nylon strips • “probes” are added (look for DQA1 variants) • DQA1 genes will hybridize with probes • Looks like a blue dot for each DQA1 gene • DQA1 frequencies are greater in a population

  29. DQA1 or HLA DQ alpha system

  30. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • 2. Polymerase Chain Reaction (PCR) [cont] • Polymarker (PM) • Type of PCR • Types 5 additional genetic markers • Used in combination with DQA1 • Can work saliva on cigarette butts • 3. Short Tandom Repeats (STR) • a. General information • Most recent method • Most successful and widely used • High discrimination and reduced period of time • STR’s are locations (loci) • Short sequences that repeat themselves • Found in great abundance

  31. STR

  32. STR

  33. Short Tandom Repeats (STR)

  34. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • 3. Short Tandom Repeats (STR) • a. General information • Usually 3 to 7 bases • Entire strand is less than 400 bases (very short) • Less susceptible to degradation • Easier to collect, especially exposed samples • ex) THO1 is A-A-T-G • (tab) amplified by PCR • (tab) separate with electrophoretic gel • (tab) count the number of THO1

  35. I. DNA: A Forensic Science Tool • F. DNA Typing (cont) • 3. Short Tandom Repeats (STR) • a. General information (cont) • THO1 (cont) • Hundreds of STR’s • The more identified, the smaller the percent of people with the same combination • (indent) multiplexing- using more than 1 STR during PCR process • (indent)(indent) Ex. STR Blue Kit, uses 3 STR’s

  36. The 13 CODIS STRs and Probability of Identities

  37. STR

  38. THO1

  39. I. DNA: A Forensic Science Tool • Developed capillary electrophoresis for STR identification • (indent) carried out in a glass column • Sexing comes from the amelogenin gene on the x and y • (indent) shorter on the x than y (6 bases) • 4. Mitochondrial DNA • a. General concepts • Second type of DNA • Written as mtDNA • Inherited solely from the mother • Found in all cells (hundreds to thousands per cell)

  40. I. DNA: A Forensic Science Tool • 4. Mitochondrial DNA • a. General concepts (cont.) • Convert sugar into ATP • Contains several loops of DNA per mitochondria • Can get DNA from relative, when no DNA exists from a person long since dead • More rigorous, time consuming, and costly • Few labs run this type of testing • Very few crimes are “allowed” to use this type (cost-benefit ratios) • mtDNA is circular (like bacterial DNA) • Two regions are “highly” variable

  41. I. DNA: A Forensic Science Tool • 4. Mitochondrial DNA • a. General concepts (cont.) • Called Hypervariable region 1 (HV1) and Hypervariable region 2 (HV2) • Several labs have created an mtDNA database • (indent) sample size isn’t large enough to have complete statistical data • 5. Combined DNA Index System • a. General concepts • Will be used to look at sex crimes against criminal databases • All 50 states have legislation to collect DNA convicted from sex-offenders

  42. I. DNA: A Forensic Science Tool • b. Combined DNA Index System • Called CODIS • Standardized 13 STR’s • Created from the 50 states’ databases

  43. Gel Electrophoresis

  44. II. Collection and Preservation of Biological Evidence • A. General Concepts • 1. Evidence must be collected in the appropriate manor • 2. Position and shape of biological material can be crucial pieces of evidence • 3. Should always assume the biological evidence is infectious • 4. Should attempt to find towels or rags, in case of hand wiping • 5. Should not put into airtight containers • Bacteria can grow and destroy evidence • Fungi as wells

  45. II. Collection and Preservation of Biological Evidence • 6. Placed in a paper bag and refrigerated • 7. Blood (dried) should be moved with a cotton swab dipped in distilled water (dH2O) • Nearby areas are swabbed as a control • 8. Use luminol to observe unseen blood stains • Does not inhibit STR’s

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