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2. Agenda: 4/28 Objective: To determine how DNA is used in forensic cases Human Genome – how people differ DNA Uses and Sources DNA Fingerprinting – Steps needed - Restriction enzymes - Gel electrophoresis - Polymerase Chain Reaction Homework: Tuesday: Lab Notebook – with Serial Dilution & Spectrophotometer Wednesday: News article – specific and detailed for credit

3. Class notebook DateAssignmentPages 4/25Processes represented in the Central Dogman 4/25Proteins- function and structure DNA Fingerprinting - Use of DNA in Forensic Cases Restriction enzymes – background & lab preparation

4. DNA fingerprinting DNA forensics Digesting a DNA sample using restriction enzymes Gel electrophoresis –Process: running gels –Data analysis Polymerase Chain Reaction –Process –Interpretation Solving the Case Paternity Case - Blacketts What we need to know: RFLP –Restriction Fragment Length Polymorphism Restriction enzymes How/why does gel electrophoresis works? How/why does PCR work? Use of informatics/statistics

5. The Human Genome The sequence of bases make up our genes. The Human Genome Project determined the order of each of these bases in all of our genes. Also found that most DNA is not coding for genes. There are many areas in which bases are repeated.

6. How many bases are there in the human genome? a)3,000 b)300,000 c)3 million d)3 billion e)3 trillion Facts & Figures about DNA

7. How many bases are there in the human genome? Facts & Figures about DNA

8. How many bases are there in the human genome? Facts & Figures about DNA a)3,000 b)300,000 c)3 million d)3 billion e)3 trillion

9. How many bases are there in the human genome? Facts & Figures about DNA 3,000,000,000

10. We are not all exactly the same – What percent of your DNA is similar to any other person in the world? Facts & Figures about DNA

11. We are not all exactly the same – What percent of your DNA is similar to any other person in the world? a)99.9% b)98% c)90% d)60% e)10% Facts & Figures about DNA

12. We are not all exactly the same – What percent of your DNA is similar to any other person in the world? Facts & Figures about DNA a)99.9% b)98% c)90% d)60% e)10%

13. We are not all exactly the same – What percent of your DNA is similar to any other person in the world? Facts & Figures about DNA 3 MILLION bases are different!

14. Forensic scientists focus on these variable regions to generate a “DNA fingerprint” for each individual Facts & Figures about DNA

15. Summary – Nuclear DNA

16. DNA Use in Forensic Cases Most are rape cases (>2 out of 3) Looking for match between evidence and suspect Must compare victim’s DNA profile Mixtures must be resolved DNA is often degraded Inhibitors to PCR are often present Challenges

17. Human Identity Testing Forensic cases -- matching suspect with evidence Paternity testing -- identifying father Historical investigations Missing persons investigations Mass disasters -- putting pieces back together Military DNA “dog tag” Convicted felon DNA databases

18. YouTube – DNA forensics – 4 videos https://www.youtube.com/watch?v=dXYztbkMXwU&list =PLC0B027FC81C82602 – 2 minute overviewhttps://www.youtube.com/watch?v=dXYztbkMXwU&list =PLC0B027FC81C82602 Includes CODIS – Story https://www.youtube.com/watch?v=VF5s1loHxx4&list=P LC0B027FC81C82602https://www.youtube.com/watch?v=VF5s1loHxx4&list=P LC0B027FC81C82602 https://www.youtube.com/watch?v=8w_VJ4G7qiw&list=P LC0B027FC81C82602https://www.youtube.com/watch?v=8w_VJ4G7qiw&list=P LC0B027FC81C82602 https://www.youtube.com/watch?v=yWKb QH2P6og&list=PLC0B027FC81C82602https://www.youtube.com/watch?v=yWKb QH2P6og&list=PLC0B027FC81C82602

19. What are some sources of DNA?

20. Sources of Biological Evidence Blood Semen Saliva Urine Hair Teeth Bone Tissue Mucus Ear Wax

21. DNA fingerprinting DNA forensics Digesting a DNA sample using restriction enzymes Gel electrophoresis –Process: running gels –Data analysis Polymerase Chain Reaction –Process –Interpretation Solving the Case Paternity Case - Blacketts What we need to know: RFLP –Restriction Fragment Length Polymorphism Restriction enzymes How/why does gel electrophoresis works? How/why does PCR work? Use of informatics/statistics

22. Cutting the DNA with Restriction Enzymes

23. DNA can be cut into smaller pieces by restriction enzymes that recognize very specific sequences of DNA. Restriction Enzyme Digest AGCTAGAATTCTTTACGCTCGGATGAATTCC ACCTATCTCC

24. DNA can be cut into smaller pieces by restriction enzymes that recognize very specific sequences of DNA. AGCT AG AATTCTTTACGCTC GGATG AATTCCACCTAT CTCC Restriction Enzyme Digest AGCTAGAATTCTTTACGCTCGGATGAATTCC ACCTATCTCC

25. Multiple Restriction Enzymes Exist for Cutting DNA EcoRI GAATTC G AATTC PstI CTGCAG CTGCA G SmaI CCCGGG CCC GGG HindIII AAGCTT A AGCTT BamI GGATCC G GATCC HaeIII GGCC GG CC

26. Separating the DNA fragments RFLP analysis

27. Visualizing the DNA Restriction Fragments 1 – Ladder to determine size (number of base pairs in each segment) 2-7 samples from suspects or victims

28. DNA Restriction Enzymes Evolved by bacteria to protect against viral DNA infection Endonucleases = cleave within DNA strands Over 3,000 known enzymes

29. Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Palindrome Restriction site Fragment 1 Fragment 2

30. 5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts

31. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

32. The DNA Digestion Reaction Restriction Buffer provides optimal conditions NaCl provides the correct ionic strength Tris-HCI provides the proper pH Mg 2+ is an enzyme co-factor

33. DNA Digestion Temperature Why incubate at 37°C? Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? Too hot = enzyme may be denatured (killed) Too cool = enzyme activity lowered, requiring longer digestion time

34. Restriction Fragment Length Polymorphism RFLP Allele 1 Allele 2 GAATTC GTTAAC GAATTC GTTAAC CTGCAG GAGCTC CGGCAG GCGCTC PstIEcoRI 123 3 Fragment 1+2 Different Base Pairs No restriction site + MA-1A-2 Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments

35. Agarose Electrophoresis Loading Electrical current carries negatively- charged DNA through gel towards positive (red) electrode Power Supply Buffer Dyes Agarose gel

36. Agarose Electrophoresis Running Agarose gel sieves DNA fragments according to size –Small fragments move farther than large fragments Power Supply Gel running

37. Analysis of Stained Gel Determine restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples

38. Molecular Weight Determination Size (bp)Distance (mm) 23,00011.0 9,40013.0 6,50015.0 4,40018.0 2,30023.0 2,00024.0 Fingerprinting Standard Curve: Semi-log

39. Polymerase Chain Reaction (PCR) PCR can make many copies in a very short period of time ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT

40. Polymerase Chain Reaction (PCR) Heat to 94°C: Denature Strands of DNA ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT

41. ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT Polymerase Chain Reaction (PCR) Cool to 55°C: Allow primers to anneal TGCGTGAA TGACTGAA

42. ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT Polymerase Chain Reaction (PCR) Heat to 72°C: New DNA strand is synthesized TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA

43. Polymerase Chain Reaction (PCR) PCR can make many copies in a very short period of time ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT

45. How do we generate a DNA fingerprint? …After amplification of the variable regions through PCR

46. FBI’s CODIS DNA Database Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1998 Links all 50 states Requires >4 RFLP markers and/or 13 core STR markers Current backlog of >600,000 samples

47. 13 CODIS Core STR Loci with Chromosomal Positions CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539D18S51 D8S1179 D3S1358 FGA VWA AMEL

48. Overview Basic – DNA Fingerprinting –Overview: 6 min. Bozeman Science

49. Use of Short Tandem Repeats Non-coding sections (do not code from proteins) Inherited from parents –Individuals have 2 copies (alleles)

50. 13 CODIS Core STR Loci with Chromosomal Positions CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539D18S51 D8S1179 D3S1358 FGA VWA AMEL

51. Short Tandem Repeats (STRs) the repeat region is variable between samples while the flanking regions where PCR primers bind are constant 7 repeats 8 repeats AATG Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

52. Short Tandem Repeats STRs are short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times Example: the 16 bp sequence of "gatagatagatagata" would represent 4 copies of the tetramer "gata". The polymorphisms (variations in DNA sequence between individuals) in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals.

53. STR and probability Today's DNA profile :: DNA Learning CenterToday's DNA profile :: DNA Learning Center http://www.dnalc.org/view/15983-Today-s- DNA-profile.html

54. STR Allele Frequencies Caucasians (N=427) Blacks (N=414) Hispanics (N=414) TH01 Marker * Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34 Number of repeats Frequency

55. 170 bp 195 bp Different primer sets produce different PCR product sizes for the same STR allele TCAT repeat unit

56. Multiplex PCR Over 10 Markers Can Be Copied at Once Sensitivities to levels less than 1 ng of DNA Ability to Handle Mixtures and Degraded Samples Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

57. ABI Prism 310 Genetic Analyzer capillary Syringe with polymer solution Autosampler tray Outlet buffer Injection electrode Inlet buffer

58. Close-up of ABI Prism 310 Sample Loading Area Autosampler Tray Sample Vials Electrode Capillary See Technology section for more information on CE

59. amelogenin D19 D3 D8 TH01 VWA D21 FGA D16 D18D2 amelogenin D19 D3 D8 TH01 VWA D21 FGA D16 D18 D2 Two different individuals DNA Size (base pairs) Results obtained in less than 5 hours with a spot of blood the size of a pinhead probability of a random match: ~1 in 3 trillion Human Identity Testing with Multiplex STRs Simultaneous Analysis of 10 STRs and Gender ID AmpFlSTR ® SGM Plus™ kit

60. STR genotyping is performed by comparison of sample data to allelic ladders Microvariant allele

61. STR Allele Frequencies Caucasians (N=427) Blacks (N=414) Hispanics (N=414) TH01 Marker * Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34 Number of repeats Frequency

62. 4 types of DNA http://learn.genetics.utah.edu/content/extras/ molgen/index.htmlhttp://learn.genetics.utah.edu/content/extras/ molgen/index.html

63. When nuclear DNA is degraded:

64. Cases OJ Simpson http://www.trutv.com/library/crime/criminal _mind/forensics/serology/5.htmlhttp://www.trutv.com/library/crime/criminal _mind/forensics/serology/5.html Australian http://www.trutv.com/library/crime/criminal _mind/forensics/serology/5.html

65. Blackett Family Paternity Case http://www.biology.arizona.edu/human_bio/ activities/blackett2/overview.html