Technology and Genetics of DNA Fingerprinting Image from:http://www.virginmedia.com/microsites/technology/slideshow/vm-tech-gallery/civilliberties/img_5.jpg

Student Focus Questions What is DNA fingerprinting? What are the building blocks of DNA fingerprinting? What is the significance of Polymorphic DNA regions? What is the purpose of PCR analysis?

What is DNA Fingerprinting? DNA Fingerprinting – Procedure that analyzes an individual’s unique collection of DNA restriction fragments, detected by electrophoresis and nucleic acid probes. Common uses for DNA Fingerprinting Forensic science tool in criminal cases Paternity cases Determine origin of nonhuman materials Botany and Zoology studies

DNA Fingerprinting and Crimes DNA fingerprinting generated world-wide attention in a murder case in 1986. DNA analysis is now an integral piece of forensic science. In the Narborough murder case, one man was proved innocent and another guilty. Image from: www.magnumphotos.com/.../F/P/8/D/LON41005.jpg First DNA results in Narborough, England case.

Genetics of DNA Fingerprinting DNA fingerprinting, (also known as typing) is based on different genomes (base pair for base pair) being different for each individual. Identical twins, however, will have identical genomes. Image from:http://www.topnews.in/health/files/Identical-Twins.jpg Identical Twin Electrophoresis Gel Image from: Stanford School of Medicine

Genetics of DNA Fingerprinting (cont.) The building blocks of DNA are the base pairs: Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). The order in which the base pairs are arranged determines the gene function. Image from:http://ghr.nlm.nih.gov/handbook/illustrations/dnastructure.jpg

Genetics of DNA Fingerprinting (cont.) Polymorphic DNA regions. These regions vary in length from individual to individual and fall into two categories: 1. Variable Number of Tandem Repeats (VNTR) and 2. Short Tandem Repeat (STR). A VNTR is a region that is variably composed of a 15-70 base pair sequence, typically repeated 5-100 times. Top chromosome includes 12 motifs (sequence of bases A, G, C, and T) while the lower chromosome has 17 motifs. The number of repeats varies by individual from 5 to more than 100. Image from: University of Michigan

Polymorphic DNA regions. (cont.) Short Tandem Repeat (STR) is similar to VNTR except that the repeated unit is only 2-4 nucleotides in length. Examination of several different VNTRs or STRs from the same individual, reveal a DNA fingerprint which is unlike anyone else’s (except for identical twins)!

Genetics of DNA Fingerprinting (cont.) DNA Fingerprinting Techniques 1. Polymerase Chain Reaction (PCR) Often samples collected are quite small. The Polymerase Chain Reaction (PCR) is a technique which copies any segment of DNA quickly and precisely. VNTR PCR process: Denaturing phase - Sample is treated with a detergent to rupture cell membranes, this frees the DNA. Heat the sample for denaturing to occur. At 94 degrees C. the DNA double helix separates (denatures) into individual strands. Image from: www.enigmadiagnostics.com/ufiles/PCRstep3.gif

Polymerase Chain Reaction (PCR) (cont.) Annealing phase - Synthetic molecules of DNA bind specifically to their complementary sequence at around 40 - 60 degrees C. This allows hybridization of the two primers to the two strands (one to each of the two strands of the target DNA sequence). Image from: www.enigmadiagnostics.com/ufiles/PCRstep3.gif

Polymerase Chain Reaction (PCR) (cont.) Extension phase – Also known as DNA synthesis, the temperature is raised to 72 degrees C. and the Taq DNA polymerase adds nucleotides to the primers to complete the synthesis of the new complementary strands. Taq polymerase extends the synthetic DNA molecules and creates new copies of each strand Image from: www.enigmadiagnostics.com/ufiles/PCRstep3.gif

Polymerase Chain Reaction (PCR) (cont.) Cycling phase – Is the combination of denaturation, annealing and extension phase one time. This process is repeated 30-40 cycles, amplifying the target sequence exponentially. Each round of PCR, which is performed on a thermal cycler (top), doubles the total quantity of DNA. Image from: Benjamin Cummings

Polymerase Chain Reaction (PCR) (cont.) Thermal Cyclers Thermal cyclers are programmed to rapidly heat, cool and maintain samples at designated temperatures for varying amounts of time. Thermal cycler with DNA samples being loaded. Image from: www.labcentraal.com/portals/14/images/Applied

Polymerase Chain Reaction (PCR) (cont.)

Short Tandem Repeat (STR) STR site 1 STR site 2 STR analysis is a technique for producing and comparing DNA fingerprints that reflect the lengths of STR sequences at specific sites in the genome. Crime scene DNA Number of short tandem repeats match Number of short tandem repeats do not match Suspect’s DNA Randomly scattered throughout the genome, STR sites contain tandem repeats of four- nucleotide sequences. In the image above, both samples have 7 repeats at STR site 1, however, different numbers (8 vs. 13) at STR site 2. Image from: Benjamin Cummings

Gel Electrophoresis Once the DNA fragments are prepared, the next step is to determine the fragment lengths. Gel electrophoresis is a tool and method which sorts macromolecules – normally proteins or nucleic acids. The first step is to load to the wells, which are located at one end of the gel. Loading DNA fragments into wells Images from: farm3.static.flickr.com/2290/2088536114_aff25...

Gel Electrophoresis (cont.) Electrophoresis equipment: Power supply, electrophoresis horizontal chambers with loaded gels.

Gel Electrophoresis (cont.) Once the wells are loaded, a negatively charged electrode is attached to the DNA-containing end of the gel and the positive electrode to the opposite end. Phosphate (PO4-) groups give DNA fragments a negative charge, which pushes them toward the positive pole. Larger DNA fragments are inhibited by dense polymer fibers, so they move slower, than shorter fragments. Mixture of DNA fragments of different sizes Longer (slower) fragments Power supply Gel Shorter (faster) fragments Completed gel Image from:Benjamin Cummings

Gel Electrophoresis (cont.) After electrophoresis, agarose gels require staining to visualize the separated DNA samples. Stain such as Ethidium Bromide is placed on the gel so the bands are visible. Gel staining with visible bands. Image from: www.thermo.com/com/CMA/Images/Image_43862.jpg

Gel Electrophoresis (cont.) Overview of DNA fingerprinting In this example, DNA from suspect 2 matches DNA collected at the crime scene. The sample from suspect 1 is not a match.

Student Review Questions Why is only a trace of DNA evidence needed for forensic analysis? What is polymorphic DNA? What are STRs, and how are they useful for DNA fingerprinting? What are VNTRs, and how are they useful for DNA fingerprinting? List the base pairs which are building blocks in DNA analysis. What are the PCR phases, and what do they do?

Student Review Questions (cont.) 7. What’s the significance of Taq DNA polymerase? 8. PCR cycling does what to the DNA fragment? 9. Proteins are macromolecules or micro- molecules? 10. Describe how DNA fingerprinting is utilized in criminal investigations.

References 1. Campbell, N., Reece, J. and Simon, E. Essential Biology, Third Edition. 2007. Benjamin Cummings, Ch. 12. 2. Can DNA Demand a Verdict? Retrieved from: http://learn.genetics.utah.edu/content/labs/gel/forensics/ 3. DNA Fingerprinting, Genetics and Crime: DNA Testing and the Courtroom. University of Michigan. Retrieved from: http://www.fathom.com/course/21701758/index.html 4. Genetics of DNA Fingerprinting Paternity Test. Retrieved from: http://images.google.com/imgres?imgurl=http://scienceprofonline.googlepages.com/DNAMicrotubeGravityWaveFlickr.jpg/DNAMicrotubeGravityWaveFlickr-full.jpg&imgrefurl=http://scienceprofonline.googlepages.com/paternitytestingusingdnafingerprinting&usg=__PVsHxX9mMO3zdyR4xc2R-8Ju6I0=&h=375&w=500&sz=23&hl=en&start=1&tbnid=EfJayvTUYKveRM:&tbnh=98&tbnw=130&prev=/images%3Fq%3DGenetics%2Bof%2BDNA%2Bfingerprinting%26gbv%3D2%26ndsp%3D20%26hl%3Den%26sa%3DG DDNA Fingerprinting, Genetics and Crime: DNA Testing and the Courtroom NA Fingerprinting, Genetics and Crime: DNA Testing and the Courtroom