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Crime Scene Investigator PCR Basics™

Crime Scene Investigator PCR Basics™. Workshop Time Line. Monday: Introduction to DNA profiling Pre-lab Quiz Tuesday: Set up PCR reactions Exercise 1 Thursday: Electrophorese of PCR products Analysis and interpretation of results Monday: Exercise 2 Tuesday: Post Test.

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Crime Scene Investigator PCR Basics™

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  1. Crime Scene Investigator PCR Basics™

  2. Workshop Time Line Monday: • Introduction to DNA profiling • Pre-lab Quiz Tuesday: • Set up PCR reactions • Exercise 1 Thursday: • Electrophorese of PCR products • Analysis and interpretation of results Monday: • Exercise 2 Tuesday: • Post Test

  3. What is DNA profiling? DNA profiling is the use of molecular genetic methods to determine the exact genotype of a DNA sample in a way that can basically distinguish one human being from another The unique genotype of each sample is called a DNA profile.

  4. Prep. for Lab—Crime Scene Investigator In Advance • Label Tubes: • 9 yellow MMP • 8 purple CS • 8 green A • 8 blue B • 8 orange C • 8 pink D • 8 LD • 8 AL (Allele Ladder) • Set Thermocycler (page 16) • Add loading Dye to Ladder • Aliquot Loading Dye 1 hour before PCR • Add primers to master mix (see page 15) • Aliquot master mix with primers (see page 16) • Aliquot CS and suspect A-D DNA (see page 16) • Get Ice for each station Before Gels (page 18-19) • Pour 8 Gels—3%, EtBr 1ul/50mL, 6 well combs • (3g/100ml) 12g/400ml • -8ul EtBr • Prepare TAE Buffer

  5. How do crime scene investigators create a DNA profile? 1. Evidence is collected at the crime scene: Blood Tissue Semen Urine Hair Teeth Saliva Bone

  6. How do crime scene investigators create a DNA profile? • 2. DNA is extracted from sources at the crime scene and from victims and suspects

  7. DNA Quantitation PCR Amplification of multiple (STR) markers Sample Genotype Determination Generation of Case Report with Probability of Random Match If match occurs, comparison of DNA profile to population databases How do crime scene investigators create a DNA profile? • 3. DNA samples are processed Sample Obtained from Crime Scene or Paternity Investigation Biology DNA Extraction Technology Separation and Detection of PCR Products (STR Alleles) Genetics Comparison of Sample Genotype to Other Sample Results

  8. 4. Crime Scene Investigators search in areas of the genome that are unique from individual to individual and are “anonymous”(control no known trait or function) The areas examined are Short Tandem Repeats or STR’s • STR region Since humans are 99.9% identical where do crime scene investigators look for differences in DNA profiles?

  9. Example of an STR: TH01 • The TH01 locus contains repeats of TCAT. • CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA • This example has 6 TCAT repeats. • There are more than 20 known TH01 alleles. • Each individual inherits 1 allele from each parent.

  10. 5’ 3’ Starting DNA Template 3’ 5’ Target DNA To determine the genotype (DNA profile) Crime Scene Investigators make billions of copies of the target sequence using PCR PCR

  11. What’s the point of PCR? • PCR, or the polymerase chain reaction, makes copies of a specific piece of DNA • PCR allows you to look at one specific piece of DNA by making copies of *only* that piece of DNA • PCR is like looking for a needle in a haystack, and then making a haystack out of the needle

  12. DNA profiling is used to determine which suspect can not be excluded from suspicion.

  13. How are suspects included or excluded from an investigation? • Suspects are included in an investigation if their DNA profile matches with genotypes found at the crime scene • Suspects can be excluded if their DNA profile does not match genotypes found at the crime scene

  14. Crime Scene Investigator PCR Basics™ProceduresOverview

  15. LaboratoryQuick Guide (You have these instructions in your packet.)

  16. Day 1 LabSet up PCR reactions • Find the PCR tubes at your station. Label them ‘CS’ for Crime Scene DNA, ‘A’ for Suspect A DNA, ‘B’ for Suspect B DNA, ‘C’ for Suspect C DNA, and ‘D’ for Suspect D DNA. • Keeping the tubes on ice, add 20 μl of Master Mix + blue primers to each tube. • Keeping the tubes on ice, add 20 µl of each DNA to the appropriately labeled tube. • Mix and put in thermal cycler • Cycle ~3 hours

  17. Day 1 Tips • Use a fresh tip every time!!! • To cool the tubes down even more, put water in the same beaker with the ice. • Do this at the beginning of the lab! • Make sure the caps of the tubes are on tight when you put them in ice! • When you put in the thermal cycler, follow the chart that Mrs. Duffy gives you exactly! • You will misplace your tubes otherwise!

  18. What is needed for PCR? Reverse primer 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Forward primer Target sequence The PCR ReactionWhat do you need? • Template (containing the STR you want to amplify for the study) • Sequence-specific primers flanking the target sequence • Nucleotides (dATP, dCTP, dGTP, dTTP) • Magnesium chloride (enzyme cofactor) • Buffer, containing salt • Taq polymerase

  19. What is happening in the PCR tube while in the thermocycler? PCR Animation http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html

  20. Heat (94oC) to denature DNA strands Cool (52oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 35 cycles The PCR ReactionHow does it work?

  21. Denaturing Template DNA 3’ 5’ Heat causes DNA strands to separate 3’ 5’ 3’ 5’ Denaturation of DNA at 94oC 5’ 3’

  22. Annealing Primers Primers anneal at 52oC • Primers bind to the template sequence • Taq polymerase recognizes 3’ end of primer + template strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Taq extendsat 72oC 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’

  23. Cycle 1 STR DNA is replicated Cycle 2 Repeat denaturing, annealing, and extending 35 cycles Cycle 3 The exact-length target product is made in the third cycle

  24. Allele ladder Mother Father Child C Child D Child E TH01 alleles To visualize PCR products Crime Scene investigators use gel electrophoresis (14) (13) (12) (11) (10) (9) (8) (7) (6) (5) (4) (3)

  25. Day 2 LabElectrophorese PCR products • Add 10 ul of Orange G Loading Dye to each PCR tube and mix • Set up gel and electrophoresis equipment • Load 20 ul of CSI allele ladder to Lane 1 • Load 20 ul of your PCR reactions in lanes 2 to 6 • Electrophorese samples • Stain gel with Fast Blast DNA Stain • Analyze results

  26. Using the digital micropipetAdd 10ul of loading dye to each microtube

  27. AgaroseElectrophoresisPlace gel in gel boxPour buffer in box until gel wells are covered.

  28. Place 20ul of samples into appropriate wells Set up electrophoresis chamber by putting top in place and connecting it to the power supply

  29. AgaroseElectrophoresisRunning Agarose gel sieves DNA fragments according to size – Small fragments move farther than large fragments Use a 3% gel to separate small fragment sizes Gel running

  30. Day 2 Tips • Use a method of loading that allows you to keep your hands steady! • When you put your gel into the gel box make sure that the wells are on the black end so that the DNA “runs to red”.

  31. Analysis of Results:Who can’t be excluded? CS A B C D AL 15 10 AL: Allele ladder CS: Crime Scene A: Suspect A B: Suspect B C: Suspect C D: Suspect D 7 BXP007 alleles 5 4 3 2 1 5-2 7-4 5-2 7-2 10-3 genotype

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