DNA

A NYC Case Example

Sexual Assault in Building Hallway

Blood-soaked victim's shirt

Case Facts: Person seen running from scene ID of person made by tenant Police arrest suspect Suspect found to have prior history of sexual assault Suspect confesses Rape kit is negative for semen

DNA Testing Results Blood stains on stairs do not match suspect Local DNA database found a match with a semen stain from another case Similar modus operandi and nearby location Suspect identified in other case DNA match with semen of other case

Did suspect have an accomplice?

Epilogue Mixtures of DNA from victim and perpetrator found in bloodstain from victim’s clothing Suspect’s DNA not found on clothing or crime scene Charges dropped against suspect

What is DNA?

What does DNA look like?

Basic Components of Nucleic Acids Phosphate Base 5’end | Phosphate Sugar—Base… 3’end Sugar

Base Pairing of DNA Strands A = T G  C T = A C  G T C A G 5’ 3’ denatured strands hybridized Hydrogen bonds Phosphate-sugar backbone

Sources of DNA Blood and blood stains Semen and semen stains Bones Teeth Hair with roots Hair shaft Saliva (containing nucleated cells) Urine Feces Debris from fingernail scrapings Muscle Tissue Cigarette butts Postage stamps Envelope sealing flaps Dandruff Fingerprints Personal Items: razor blade, chewing gum, toothbrush, lipstick stains Adopted from Butler, J.M. (2001) Forensic DNA Typing, Table 3.1, ©Academic Press

DNA BASICS Genomic DNA as hereditary material -found in the nucleus of the cell (nuclear DNA) Determines physical characteristics of people Half the DNA is maternal/half the DNA is paternal

DNA BASICS Genomic DNA is found in every nucleic cell in the body Genomic DNA is the same in all cells Genomic DNA does not change throughout one’s lifetime

DNA BASICS No two people (except identical twins) have exactly the same DNA Mostly the same between people, but small regions vary between individuals Less than 0.1% of DNA is unique

What we can’t tell from forensic DNA testing….. Race- (now we can!) Age Diseases (unless genetic!) How it got there

How is DNA Used in Forensic Analysis? Entire genome is not examined “Polymorphic” sequences of the genome are examined Polymorphic areas are present on “loci” - Two “alleles” at each loci

Types of DNA Polymorphisms Length Polymorphism ----(AATG)(AATG)(AATG)----- 3 repeats -----(AATG)(AATG)---- 2 repeats

Variable Number of Tandem Repeats (VNTR) Repetitive DNA in human genome (20-30%) Satellite DNA Minisatellite (extreme variability) DNA 10-100 base pair repeats Polymorphism based on number of tandem repeats

STRs (microsatellite DNA) Preferred genetic marker in forensic DNA testing Tetranucleotide repeat sequence CODIS (combined offender DNA indexing system) loci

Brief History of DNA Typing 1980 - Ray White describes first polymorphic RFLP marker 1985 - Alec Jeffreys discovers multilocus VNTR probes 1985 - first paper on PCR 1988 - FBI starts DNA casework 1991 - first STR paper 1995 - FSS starts UK DNA database 1998 - FBI launches CODIS database

DNA processing from scene to court

DNA Extraction Organic Method INCUBATE (56 oC) Centrifuge Blood stain SDS, DTT, EDTA and proteinase K Phenol, chloroform, isoamyl alcohol VORTEX Centrifuge TRANSFER aqueous (upper) phase to new tube TE buffer CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge QUANTITATE DNA PERFORM PCR

PCR Polymerase Chain Reaction=Simulated Natural DNA replication

DNA Replication http://www.johnkyrk.com/DNAreplication.html

PCR http://www.dnalc.org/ddnalc/resources/animations.html

Thermal Cycling Temperatures 94 oC 94 oC 94 oC 94 oC Single Cycle 72 oC 72 oC 72 oC Temperature 60 oC 60 oC 60 oC Time Typically 25-35 cycles performed during PCR 94oC - Denaturation 60oC - Annealing 72oC - Extension

PCR

PCR

Number of Target Molecules Created 1   2 3 4 5 8 6 16 7 32 64 9 128 10 256 11 512 12 1024 13 2048 14 4096 15 8192 16,384 17 32,768 18 65,536 19 131,072 20 262,144 21 524,288 22 1,048,576 23 2,097,152 24 4,194,304 25 8,388,608 26 16,777,216 27 33,544,432 28 67,108,864 29 134,217,728 30 268,435,456 31 536,870,912 1,073,741,824 Cycle Number Number of Double-stranded Target Molecules

Advantages of PCR Need very small amounts of biological material Minute amounts of DNA template may be used (i.e., 3 uL whole blood, 2-3 mm bloodstain cutting) Can be used with forensic samples DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification. Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used.

DNA Replicated via PCR, Now what? Electrophoresis and Southern Blotting http://www.pbs.org/wgbh/nova/sheppard/labwave.html DNA to convict the guilty and exonerate the innocent