Wright State University CORE Scholar Biological Sciences Faculty Publications Biological Sciences 12-2012 Generating Forensic DNA Profiles Dan E. Krane Wright State University - Main Campus, dan.krane@wright.edu Follow this and additional works at: http://corescholar.libraries.wright.edu/biology Part of the Biology Commons, Medical Sciences Commons, and the Systems Biology Commons Repository Citation Krane, D. E. (2012). Generating Forensic DNA Profiles.. http://corescholar.libraries.wright.edu/biology/247 This Presentation is brought to you for free and open access by the Biological Sciences at CORE Scholar. It has been accepted for inclusion in Biological Sciences Faculty Publications by an authorized administrator of CORE Scholar. For more information, please contact corescholar@www.libraries.wright.edu.
Generating forensic DNA profiles Dan E. Krane, Wright State University, Dayton, OH Forensic DNA Profiling Video Series Forensic Bioinformatics (www.bioforensics.com)
Three generations of DNA testing RFLP AUTORAD Allele = BAND DQ-alpha TEST STRIP Allele = BLUE DOT Automated STR ELECTROPHEROGRAM Allele = PEAK
Two additional DNA tests Mitochondrial DNA mtdna sequence Sensitive but not discriminating Y-STRs Useful with mixtures Paternally inherited
DNA content of biological samples Trillions of cells Roughly 100 cells Each cell contains 6 to 7 pg of DNA DNA profiling kits generally recommend using between 500 and 1,000 pg of template DNA That works out to roughly 100 to 200 cells
What is a picogram? 1 gram = 1/4 th of a packet of sugar 1 milligram = a single crystal of sugar 1 nanogram = one 1000 th of a crystal of sugar 1 picogram = one billionth of a gram
What is a microliter? 1 liter = half of a bottle of a soft drink 1 milliliter = 1000 th of a liter (about a thimble full) 100 microliters = one drop 1 microliter = one millionth of a liter
Basic terminology: Genetics DNA Polymorphism ( many forms ) Regions of DNA which differ from person to person Locus (plural = loci) Site or location on a chromosome Allele Different variants which can exist at a locus
Basic terminology: Technology Amplification or PCR (Polymerase Chain Reaction) A technique for replicating DNA in the laboratory ( molecular Xeroxing ) Region to be amplified defined by primers Electrophoresis A technique for separating molecules according to their size
Automated STR Test
Crime Scene Samples & Reference Samples Extract and purify DNA Differential extraction in sex assault cases attempts to isolate DNA from sperm cells
Extract and Purify DNA Add primers and other reagents
Setting up an amplification Pipettors are used to transfer microliter quantities of liquids Final reaction volumes are typically 10 or 20 microliters There are no good visual clues that a solution contains DNA or that a reaction is proceeding correctly
PCR Amplification DNA regions flanked by primers are amplified
PCR Amplification Targeted regions are doubled with each round of amplification. Instead of needle in a haystack, after 28 rounds of amplification there is a needle-stack with a piece of hay. Amplified DNA fragments are fluorescently labeled.
The ABI 310 Genetic Analyzer
ABI 310 Genetic Analyzer: Capillary Electrophoresis DNA pulled towards the positive electrode DNA separated out by size: Large DNA moves slowly Small DNA moves faster Color of STR detected and recorded as it passes the detector Detector Window
Profiler Plus: Raw data
Profiler Plus DNA profile
Reading an electropherogram Peaks correspond to alleles D3 vwa BLUE FGA Amelogenin XX = female XY = male Red = ROX size standard Electropherogram Amelogenin 75 100 139 150 160 D8 D21 D18 GREEN D5 D13 D7 YELLOW 200 245 300 bps RED
Reading an electropherogram NUMBER OF PEAKS 1 peak = homozygous 2 peaks = heterozygous 3 or more peaks = mixed sample (?) POSITION OF PEAK Smaller alleles on left Larger alleles on right HEIGHT OF PEAK Proportional to amount of allele S M A L L L A R G E
Profiler Plus D3S1358 vwa FGA AMEL D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820
SGM+ D3S1358 vwa D16S539 D2S1338 AMEL D8S1179 D21S11 D18S51 D19S433 THO1 FGA
Profiler Plus DNA profile
What weight should be given to a DNA profile match? Do they have the same source? Is the match a coincidence? Has an error occurred?
Generating forensic DNA profiles Dan E. Krane, Wright State University, Dayton, OH Forensic DNA Profiling Video Series Forensic Bioinformatics (www.bioforensics.com)