1. Chapter 13 Forensic 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

5. 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

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

7. 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

8. DNA Polymerase: Unzipping DNA

10. 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

11. Recombinant DNA

13. Electro-Gel

14. Electrophoresis

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

17.  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

18. RFLP process from start to finish

19. RFLP

20. Restriction Fragment Length Polymorphism (RFLP)

21. RFLP: Determine the relationships

22. 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.

23. 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

24. PCR

25. 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

26. 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

27. PCR

29. 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

30. DQA1 or HLA DQ alpha system

31. 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

32. STR

34. Short Tandom Repeats (STR)

35. 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

36. 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

37. The 13 CODIS STRs and Probability of Identities STRAfrican American U.S. Caucasian D3S13580.0970.080 vWA0.0740.068 FGA0.0360.041 TH010.1140.080 TPOX0.0910.207 CSF1PO0.0790.128 D5S8180.1210.166 D13S3170.1390.081 D7S8200.0870.067 D8S11790.0800.069 D21S110.0420.041 D18S510.032 D16S5390.0760.091

38. STR

39. THO1

40. 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)

41. 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

42. 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

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

44. Gel Electrophoresis

45. 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

46. 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 (dH 2 O)  Nearby areas are swabbed as a control 8. Use luminol to observe unseen blood stains  Does not inhibit STR’s