Generating forensic DNA profiles Dan E Krane Wright

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 mt. DNA 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 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 BLUEFGA D 3 v. WA Amelogenin XX = female XY = male Red = ROX size standard Electropherogram D 8 D 21 GREEN D 18 Amelogenin D 5 D 13 D 7 YELLOW 75 100 139 150 160 200 RED 245 300 bps

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 D 3 S 1358 AMEL v. WA D 8 S 1179 D 5 S 818 FGA D 21 S 11 D 13 S 317 D 18 S 51 D 7 S 820

SGM+ D 3 S 1358 AMEL D 19 S 433 v. WA D 16 S 539 D 21 S 11 D 8 S 1179 THO 1 D 2 S 1338 D 18 S 51 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)