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I. TEACHER NOTES & GUIDELINES

TITLE OF LAB: DNA Profiling using Polymerase Chain Reaction (PCR)

DEVELOPERS OF LAB: David Grover JD824, Henry Johnson JD896, Daniel Lynn JD857, Patricia Nardone JD556, Kathy Poncelet JD521, Paul Kenny JD725, Anna Forster JD885, David Lewis JD854, Linda Lund JD857, Elizabeth Cuesta JD712 Thereza Lobo JD855, David Moss JD594, Julie Middleton JD570, Christine Ansorge JD818, Dana Laffin-Rebecca JD529.

OVERVIEW OF LAB:

DESCRIPTION
The lab simulates the use of DNA in forensic investigations. Students are introduced to the concepts of Polymerase Chain Reaction (PCR) and DNA fingerprinting. The DNA profiles of two suspects are compared with evidence DNA after being electrophoresed on an agarose gel and stained with Carolina BLU stain.

CURRICULUM CONSIDERATIONS
This lab is appropriate for use in studying the following units: molecular genetics, biochemistry, tools of biology and bioethical issues. It can be used with Regents and AP Biology classes.

REAL WORLD APPLICATIONS
The students will have a better understanding of what is being done when investigators are gathering and using forensic evidence.

SAFETY CONSIDERATIONS
No eating or drinking in lab. Wear safety goggles. Wear lab apron. Avoid contact with stains. Wash hands before leaving lab.

BACKGROUND INFORMATION


A. SCIENTIFIC VIEWPOINT
Gel electrophoresis is an important molecular biology tool. Deoxyribonucleic acid (DNA) sequencing, fingerprinting or "profiling" and genetic engineering are based on this technique. Gel electrophoresis separates these DNA fragments by their size or molecular mass. The resulting gel pattern differs when the sequence of nucleotides in the DNA strands is significantly differs between samples.

No two people, except identical twins, have identical DNA. Therefore, a comparison of the gel patterns of DNA from known samples with an unknown sample will allow tentative identification of the unknown. Like many types of forensic evidence, DNA analysis is strongest when used to eliminate. However, it can also be used to incriminate by assigning probabilities of random matches between the evidence and a suspect.

While the DNA from two different individuals is over 99% similar, we can use PCR and electrophoresis to analyze the differences found in the small fraction that is different. One type of DNA that falls into this category is called VNTRs or Variable Number Tandem Repeat. VNTRs are sequences where a small number of basepairs are repeated a different number of times between any two people. For example, Suspect 1 may have the sequence GCG repeated three times (GCGGCGGCG) while Suspect 2 may have GCG only once at the same DNA location. A typical number of repeats is16 to 18. The number of repeats present will change the size of that particular piece of DNA. One way to analyze these PCR products is gel electrophoresis, because electrophoresis sorts the DNA according to its molecular weight.

PCR is used to copy or amplify specific stretches of DNA such as VNTRs. PCR consists of repeating the following three steps ten to thirty times. The three steps of PCR are 1) Denature, 2) Anneal, and 3) Synthesis. This artificial process is based on how DNA is replicated in the cell.

With PCR, we are looking at one gene at a time. However, because humans are diploid, each person has two copies of each gene - one from the mother and one from the father. Therefore, each person should show one or two bands for each PCR reaction.

The DNA provided here is similar to the type of products PCR would produce. By comparing the pattern of the suspects' DNA with the evidence DNA, we can make conclusions about whether a suspect should be included or excluded as the potential perpetrator of the crime.

B. COMMON MISCONCEPTIONS
DNA is only found in somatic cells
DNA forensics can "prove" that someone is guilty
All molecules of DNA have the same sequence of nucleotides
A macroscopic sample of DNA is required to make use of gel electrophoresis
This technology only applies to use in criminal investigations
DNA fingerprinting involves actual fingerprints
DNA can only be replicated within a cell
The PCR technique can copy an entire DNA code

EQUIPMENT/MATERIALS PROVIDED BY SOTM Suspect X-1 DNA Suspect X-2 DNA Evidence 1 DNA Evidence 2 DNA Suspect Y-1 DNA Suspect Y- 2 DNA Power supply Gel electrophoresis apparatus Microcapillary tubes & pistons TBE buffer Staining trays Plastic spatula DNA Fingerpr. Educ. Res CarolinaBlu stain Microcentrifuge and tubes Kimwipes Gloves Light box Agarose gel, tubes or precast Loading Dye Petri Dishes with practice gels ** If notified in advance, SOTM will provide pre-poured agarose gels.

PROVIDED LOCALLY Goggles Lab apron Plastic garbage bags, small Distilled or deionized water Plastic wrap Microwave oven Oven mitt Hot plate + beaker

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