JS 113: Introduction to DNA Typing

1. JS 113: Introduction to DNA Typing • Pre class activities • Quiz • Schedule update • Announcements and Assignments • II. Learning Objectives • Understand why scientists study DNA. Applications of forensic DNA • Introduction to DNA- Definition and Inheritance review • DNA structure • Be able to draw DNA structure – Base Pairing AT, GC • Understand DNA replication • Summary of Introduction to DNA • d. Overview of Methods used in Forensic DNA typing • Screening • Extraction • Quantification • e. Be able to define: cell, nucleus, chromosome, • alleles, homozygous vs heterozygous • Be able to draw a Punnett Square to illustrate allele inheritance

2. Schedules, Announcements and Assignments • Schedule • Week 15 DNA continued: Questioned Documents and Computer Forensics • 11/26 DNA continued C13 • 11/28 Questioned Documents and ComputersC16,17 • Lab activity Questioned Documents • Week 16 Legal and Ethical Issues in Forensic Science, Court Testimony • 12/3 Ethical Issues in Forensic Science: • 12/5 Countering Chaos: Logic, Ethics, and the Criminal Justice System • Week 17 The future and Course Review C19 • 12/10 Student Led Final Review- Last day of class • Announcements • All completed notebooks due –Monday 12/10 • All assignments and extra credit due Monday 12/3 • FINAL EXAM : Monday December 17 1215-1430 • Required Assignment • Read Chapters 16 and 17 • Read Code of Ethics- www.cacnews.org found in the handbook online • Extra credit assignment- Up to 5 points • Find and read up to 5 articles on forensic science from Sept 2007 to present • Write a 500 word summary and 3 Q and 3 A on the articles • Submit articles, summaries and questions by 12/3

3. Summary 1 • Why study DNA • Law enforcement, evolution, agricultural, and human applications-medical diagnostics • DNA Biology and Genetics • DNA is contained in cells –the basic unit of life • Found in nuclei, mitochondria and chloroplasts • Organized in chromosomes. Located at positions called loci and come in different forms or alleles. • Homozygous if the same, heterozygous if different • Alleles segregate independently and assort randomly when on different chromosomes. Random assortment is desired for forensic DNA loci. • DNA Function and Structure • DeoxyriboNucleic Acid : blueprints of life Replication, Information storage and mutation RIM • Central Dogma DNA------->RNA------>protein transcription translation

4. Summary 2 • DNA Structure and Function continued: • Bases of DNA are Adenine, Guanine, Cytosine and Thymine- Asian Guys Can Teach: AGCT • Base pairing is A to T and G to C- DNA is where its AT • Sequence of Bases Store information- Like the sequence of numbers in a Phone Number • Nucleotides are the building blocks (dNTPs) themselves made of phosphate base and sugar= PBS- The only station Sierra and Gabriel can watch • DNA base pairs- DNA velcro (David Letterman • DNA Replication • Semi-conservative- Half republican (old) /half democrat (new) • Template directed with base pairing (AT, GC) • 5 required ingredients- primer, template, Mg, dntps, DNA polymerase (PTMDD)

5. Steps to Sample Processing • Screening- The Art- Presumptive and confirmatory tests- blood, semen and saliva • DNA Extraction- Many types • organic phenol/chloroform • Chelex • FTA paper • Silica based extractions • DNA Quantitation (yield gels, slot blot, real time PCR) • PCR Amplification • Separation/Detection • Genotype Determination • Interpretation- Report Writing- Court testimony

6. Steps in Forensic DNA typing • (Figure 6.1 Rudin and Inman 2001) Evaluation- Is it there? 1. Start with biological sample 2. Screen- blood? Semen? Saliva, human? Extraction- Get and clean DNA 3. Open cells  Get DNA 4. Methods to get DNA and purify DNA Quantify- Determine quality and quantity? 5. Quantify- How good and how much did you get? Type to determine and compare alleles 6. RFLP vs PCR 7. Determine alleles and compare DNA types Or alleles present in samples and references Interpretation of Results

7. Review: DNA is organized • inside the cell nucleus and mitochondria

8. DNA Extraction • After screening tests are performed, a spot of the material containing the biological sample is cut and placed into a tube. • In one type of extraction method (organic), heat and chemicals are added, and protein is removed. • Then the pure DNA is recovered by filtration in which the non-DNA material goes through a sieve. • (analogous to a collection of your pasta in a colander)

9. Female cell Differential Extraction MethodFor Sexual Assault Evidence Spermatozoa • Isolation of DNA from mixtures of cells in sexual assault evidence • Based on differences in cell membranes • Spermatozoa membranes have special cross links (sulphur-sulphur bonds) • These membranes are quite resistant to opening. • Vaginal epithelial cells do not contain these membranes and are more easily broken open Sperm and v cell mixture Lysis- open v cell extract Female DNA Female DNA Lysis- open sperm extract Male DNA Male DNA

10. Quantification of DNA • Following extraction, the next step is to determine the quantity of the DNA • DNA typing methods RFLP and PCR require different amounts and different quality of DNA. • RFLP typically required 50ng. PCR typically requires less than 0.5ng to 1 ng: 100 times less!

11. Quantification of DNA using Gel Electrophoresis (-) L K K – u u u • Total DNA can be quantified by running the samples in a gel. • Typically, gels are made up of agarose (a carbohydrate from seaweed). • Known DNA quantities are included • Samples are then subject to an electric current and is called electrophoresis. • DNA is negatively charged and will migrate toward the positive electrode… • Comparisons of the results are done visually or with computer software to determine the amount of DNA in the unknown sample. wells Intact DNA Degraded DNA (+) Direction of DNA fragment movement Smaller fragments move faster and are found Near the bottom of the gel

12. Slot Blot quantification :DNA-DNA HybridizationDNA is where it’s AT • DNA samples may contain non human DNA • In order to quantify the amount of human DNA in a sample, a human specific test is required • One such test is DNA-DNA hybridization using a human specific probe: D17Z1

13. Slot blot hybridization • Like in yield gels, known amounts of DNA (human) are included • DNA hybridization of D17Z1 will occur only if the sample contains human DNA • Detection of the hybridized fragments is done using an enzyme linked assay- yielding light or color

14. Comparison of Methods used for DNA Quantification Method Ease Cost Sensitivity Result UV Spectrophotometry +++++ + ++ Total DNA Yield Gel electrophoresis +++ + + Int vs deg DNA Slot Blot ++ ++ +++ Human DNA Yield Gel blot + ++ +++ Int. vs. deg human DNA Pico-green microtitre plate ++++ ++ ++++ Total DNA Alu Quant +++ +++ ++++ Human DNA Real time PCR assays +++ +++ +++++ Human DNA

15. DNA Methods 1) Extract 2) Quantitate 3) Distinguish Size Content Restriction Fragment Length Polymorphisms (RFLP) Polymerase Chain Reaction (PCR)

16. The base sequence can exhibit differences in length and content between individuals. Beyonce... AAAGAAAGAAGAAAC... Nelly Furtado... AAAGAAAGAAGA... Fergie ... AAAGAAAGAAGT... Kelly Clarkson... AAAGAAAGAAGA... Britney Spears ... AAAGAAAGAA... Christina Aguilera... AAAGAAAGAT... Eminim ... AAAGAAAGC... NSYNC ... AAAGAAAGT... Boyz to Men ... AAAGAAAG… • Although different between individuals* DNA is identical in every cell of an individuals body** Some exceptions*identical twins**diseased individuals, mtDNA (sport analogs)

19. RFLP Role Playing 1. Students are assigned a base. 2. Sequence is provided with a restriction site 5’GGCC3’ at the end. 3. A person acts as the restriction enzyme and cuts the strand. 4. The gel (classroom) is loaded, power supply is turned on (lights) and fragments are asked to slink through the class toward the front (+) end. 5. Power is stopped and we visualize the difference in migration of the short and long fragment

20. Polymerase Chain Reaction: PCR is simply repeated rounds of DNA replication PCR based systems are rapid, require less material than RFLP and less time for typing • Molecular xeroxing • Calvin and Hobbes example

21. P-P- OH • DNA Polymerase catalyzes the template directed (A-T, G-C), incorporation of dNTPs (PP is released) forming a 3’-5’ phosphodiester linkage • Direction of synthesis 5’3’ using primer 3’OH to attach incoming nucleotide 5-P’ Primer 3-OH’ 3-OH’ dNTP Template Mg++ DNA polymerase 5-P’

22. Template- DNA from blood etc. 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ PCR is simply repeated rounds of DNA replication Step 1: Denature Separate H bonds with heat at 95C 95C 3’ 5’ 55C 3’ 5’ Step 2: Anneal Primers bind at lower temp 55C 5’ 3’ 3’ 5’ 5’ 3’ 72C Step 3: Extend Taq polymerase extends primer 3’OH at 72C (dNTPs and Mg++) Step 4: Repeated 28-30 rounds of D, A, E

23. Cycle Number Number of Double-stranded Target Molecules 1 0 2 0 3 2 4 4 5 8 6 16 7 32 8 64 9 128 10 256 11 512 12 1024 13 2048 14 4096 15 8192 16 16,384 17 32,768 18 65,536 19 131,072 20 262,144 21 524,288 22 1,048,576 23 2,097,152 24 4,194,304 25 8,388,608 26 16,777,216 27 33,544,432 28 67,108,864 29 134,217,728 30 268,435,456 31 536,870,912 32 1,073,741,824 PCR Number of Target Molecules Created • Bank account paying 100% interest every 5 minutes • Swimming pool - 10 drops

24. Relative power of tests • Test type time power • RFLP-VNTR weeks +++ * • PCR: • DQAlpha- macroarray 1 day + • PM - macroarray 1 day ++ • D1S80 - gel- VNTR 2 days ++ • STRs - gel,CE, arrays 2 days +++ • mtDNA - gel, CE, arrays 2 days + • ySTRs - gel, CE 2 days + • alu -gel, CE, arrays 2 days ++ • * not useful on degraded DNA

25. DNA Chant Review The subject of our class today Is simply stated DNA Sugar-Phosphate backbone chains Hold the base pairs here’s their names Chorus: AT- AT GC- GC ATGC, ATGC (together) RFLP holy graile Put bad guys away in jail PCR can lend a hand Amplifying those weak bands ---------------->Chorus Blood, saliva, semen too, Can be used as crucial clues Fingernails and skin and hair DNA is everywhere --------------->Chorus

26. Summary 1 • Why study DNA • Law enforcement, evolution, agricultural, and human applications-medical diagnostics • DNA Biology and Genetics • DNA is contained in cells –the basic unit of life • Found in nuclei, mitochondria and chloroplasts • Organized in chromosomes. Located at positions called loci and come in different forms or alleles. • Homozygous if the same, heterozygous if different • Alleles segregate independently and assort randomly when on different chromosomes. Random assortment is desired for forensic DNA loci. • DNA Function and Structure • DeoxyriboNucleic Acid : blueprints of life Replication, Information storage and mutation RIM • Central Dogma DNA------->RNA------>protein transcription translation

27. Summary 2 • DNA Structure and Function continued: • Bases are Adenine, Guanine, Cytosine and Thymine- Asian Guys Can Teach: AGCT • Base pairing is A to T and G to C- DNA is where its AT • Sequence of Bases Store information- Like the sequence of numbers in a Phone Number • Sides of the ladder are Sugar-Phosphate backbones • Nucleotides are the building blocks (dNTPs) themselves made of phosphate base and sugar= PBS- The only station Sierra and Gabriel can watch • DNA base pairs- DNA velcro (David Letterman • DNA Replication • Semi-conservative- Half republican (old) /half democrat (new) • Template directed with base pairing (AT, GC) • 5 required ingredients of PCR - primer, template, Mg, dntps, DNA polymerase (PTMDD)

28. Summary 3 • Steps in forensic DNA typing are – Evaluation, Extraction, Quantification, Typing and Interpretation • 1) Evaluation- Is it there? Screen- blood? Semen? Saliva, human? • 2) Extraction- Get and clean DNA • Open cells -Get DNA • Organic, Chelex and FTA extractions • Quantify- Determine quality and quantity? • How good and how much did you get? • Yield Gels, Slot Blots, Real time PCR assays