Crime Scene Investigator PCR Basics™

Why Teach Crime Scene Investigator PCR Basics™ Kit ? Exciting real-world connections Tangible results Statistical Analysis Directly applicable to the curriculum

Workshop Time Line Introduction to DNA profiling Set up PCR reactions Electrophorese PCR products Analysis and interpretation of results Main issue is the 3 hours needed to run a PCR so inevitably make it more than one session, however PCR products can be stored in the fridge for a week or so.

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.

How do crime scene investigators create a DNA profile? Evidence is collected at the crime scene: Blood Tissue Semen Urine Hair Teeth Saliva Bone What kinds of evidence contain DNA?

How do crime scene investigators create a DNA profile? DNA is extracted from sources at the crime scene and from victim and suspects Where do you find DNA in cells?

Since humans are 99.9% identical where do crime scene investigators look for differences in DNA profiles? 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 These STR sequences do NOT code for anything, i.e. they are NOT genes.

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 I hate the analogy used here. Feel free to substitute something else, and then let me know what you used! The point here is to give a very general summary of PCR.

Crime Scene Investigator PCR Basics™ Procedures Overview Main pre lesson tasks are aliquoting all the components for the class.

Using the digital micropipet We were trying to get a video of how to use pipets…

Laboratory Quick Guide This is what Melissa and I have already done in order to complete the PCR stage in advance of today. Have a read through the documentation and the associated questions.

The PCR Reaction What do you need? What is needed for PCR? The PCR Reaction What 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 5’ 3’ Forward primer Reverse primer Target sequence

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 A good place to show the flash animation of PCR.

The PCR Reaction How does it work? 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

Denaturing Template DNA 3’ 5’ Heat causes DNA strands to separate 3’ 5’ 5’ 3’ Denaturation of DNA at 94oC In our cells, enzymes (helicases) accomplish the ‘denaturation’ step. 3’ 5’

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

TH01 alleles Allele ladder To visualize PCR products Crime Scene investigators use gel electrophoresis Mother Father Child C Child D Child E (14) (13) (12) (11) (10) (9) (8) (7) 2 points to this slide: A graphic visualization of what the PCR products can look like. To illustrate the point that in addition to crime scene investigations, STR DNA profiling can also be used to determine familial relationships. The children of any two parents will have a combination of alleles provided by the parents. Check out the manual on page 12 for more details. (6) (5) (4) (3)

Electrophorese 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 (approx 30-40mins) Stain gel with Fast Blast DNA Stain Analyze results

Agarose Electrophoresis Place gel in gel box Pour buffer in box until gel wells are covered.

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

Extensions and time fillers Make sure you have a read of the questions associated with each lesson

Statistics of Chance: M&M Locus 6 Possible Alleles: Green Red Yellow Blue Brown Orange

Frequency of any M&M genotype Probabilities One allele from each parent means 2 copies of gene/locus 1 6 1 6 1 36 X = Frequency of any M&M genotype

Probabilities = = 1 6 5 36 25 Locus M&M 6 alleles Jolly Rancher Mike & Ikes x = Probabilities Jolly Rancher: 5 alleles Mike & Ikes: 5 alleles 1 22,500 = Chance an individual has a given genotype 1 506,250,000 = Chance 2 people have the same genotype

Who can’t we exclude from the pool of suspects?

Real STR analysis Four different fluorescent tags have been used to identify 7 amplified loci Allele ladders are indicated by arrows The big gel is an example of what the real analytic gel looks like. The pink frame and expanded view are what the kit is mimicking – we’re analysing one part of a much more complex gel. The graphic gel is a representation of results (not the actual results).

Analysis of Results: Who can’t be excluded? CS A B C D 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 Which suspect genotype matches that found at the crime scene? Which suspect can’t be excluded? 5-2 7-4 5-2 7-2 10-3 genotype

Other kits pGLO Bacterial Transformation (genetic engineering) ELISA to track a disease (antigen or antibody detection) DNA necklaces (DNA extraction from cheek cells) Protein fingerprinting to detemine evolutionary relationships between species

This is what the results ACTUALLY look like… except for the fingerprint.

DNA I made some DNA! I just learned how today! You take some adenine And add some cytosine Thymine and guanine, too Are added to the brew Then they’re connected to Long sugar molecules. And I can hardly wait For it to replicate First it’s unravelling And then assem-bl-ing.   I’ll take my DNA And find a mate today I’ll make genetically A brand new family tree! My baby’s DNA It will display some day A handsome phenotype And perfect genotype. My children’s DNA It will create some day Grandchildren that all be Looking alot like me!