Intro and History Forensic DNA Analysis

1900-1985 (pre-DNA years)

-ABO blood typing: first genetic evidence used in court

-after 1915 increasingly used in forensic/paternity

-greater for exclusion, poor for inclusion (80% type O or A, low power of discrimination)

Discriminating Power

-the probability that a method can differentiate between two distinct samples chosen at random from a population

random match probability

-probability of matching someone else by random chance

functional protein varient

-mutated gene

-abnormal protein or absent protein

isoenzyme

-enzymes that differ in amino acid sequence but catalyze the same chemical reaction

forensic protein profiling

-power of discrimination can be one in several hundred if using multiple

-proteins are not as variable and not as stable as DNA

-pl is the pH at which the protein is electrically neutral

forensic protein profiling advantage

-improved power of discrimination over blood group typing

forensic protein profiling limitations

-poor power discrimination (~1 in 100) even with multiple systems

-poor sensitivity

-proteins are not always stable in forensic stains or found in every sample

sensitivity

-refers to the probability of a positive test, conditioned on truly being positive

First decade of forensic DNA typing

-1980: Arlene Wyman and Ray White = Restriction Fragment Length Polymorphism (RFLP)

-multiallelic: there’s a locus with a lot of variability

-its passed down through multiple generations

A brief History of Forensic DNA typing

-1985: Alec Jeffreys RFLP techniques used in a forensic case

-previously used in paternity and immigration

-first scientifically accepted forensic DNA analysis method in the United States

-now replaced with newer techniques

The principles of RFLP testing

-cut the DNA with “biological scissors” (Restriction enzymes)

-Separate Fragments of differing Length by gel electrophoresis

-Detect length-based differences (Polymorphisms) in DNA fragments of interest

difference btw microsatellite and minisatellite

-microsatellite: 1-9 bp

-minisatellite: 10-100 bp

-macrosatellite: >100 bp

tandem repeat variation occurs

-due to strand slippage during replication

restriction digest

-cutting DNA into small, variably sized, pieces with restriction enzymes called restriction endonucleases

-restriction enzyme: bacterial protein that cuts foreign DNA

-2500 restriction enzymes have been described

Restriction Enzyme Cut Sites

-Haelll: 4 base cutter, cuts on average every ~256 bases

-Hinfl: 5 base cutter, cuts on average every ~1024 bases

-Pstl: 6 base cutter, cuts on average every ~4096 bases

-when different cut sizes used they can’t be compared

Separating DNA fragments after a restriction digest with gel electrophoresis

-an agarose gel is porous allowing movement of DNA fragments

-opposites attract

-DNA has overall negative charge

-DNA moves from cathode (-) to anode (+)

-larger DNA fragments quickly get stuck in the porous gel

-shorter fragments travel a longer distance towards the anode

VNTR’s

Variable Number Tandem Repeats

-only DNA fragments containing a complementary sequence to the probe are detected

multi-locus probe

-complex patterns

-originally developed by alec jeffreys

Single-locus probe

-better for forensic samples containing mixtures

Polymerase Chain Reaction (PCR)

-developed by kary mullis in 1983

-allowed testing of partially degraded and small samples

-shortened analysis times from weeks to days

-increased ability to use DNA evidence

PCR steps

2.) Denaturing: expose DNA to raised temperatures and the hydrogen bonds between base pairs breaks and the 2 strands of the DNA molecule separate

3.) annealing: lower temperature and primers will attach through complimentary base pairing

4.) extending: raise the temperature, DNA polymerase extends the new strand of DNA from the primers using deoxynucleotides (dNTPs)

5.) repeat

real time quantitative PCR (RT-qPCR)

-determines the amount of amplifiable DNA

-reflects both quantity and quality

-analysis: cycle to cycle change in fluorescence

-more DNA = more fluorescence

Real time qPCR C_T values

-C_T: cycle threshold: number of PCR cycles for fluorescence to signal threshold

-inverse relationship between C_T value and the amount of DNA in the sample

PCR is not the whole answer

-can only duplicate smaller fragments (1k-2k bp)

-RFLP often results in bigger fragments

-6 to 100 nucleotides that repeat 2 to 100s

-instead use STRs: up to 7 nucleotides, repeated 5 to 100 times