EXAM 2 FORENSICS

RFU

Relative fluorescence Units

The higher the peak

The more fragments

The grey bars

Loci

STR polymorphism

Length, separated using electrophoresis by size and charge

-PCR amplification of specific target loci

Tagged w/ fluorescent marker

Allele with one peak

Homozygous, tall bc two alleles sit on top of eachother. Same aloe from mom and dad

Full profile of individual

Write the genotypes from each loci

3 alleles at loci

Contamination or genetic disorder oR MIXTURES

Biological stochastic

stutter - when polymerase gets lazy, strand slippage, stops amplifying, low amounts of DNA causes more stutter

Mixtures - More than 1 person

Drop out - false homozygosity, when polymerase amplifies one allele better than the other

Technical stochastic

Voltage spikes, air bubbles, dye blobs

If sample from suspect matches crime scene evidence then

a. Suspect deposited the sample

b. Suspect has profile by chance

c. Result is laboratory error

DNA Workflow

Extraction - bc we want nucleus safely sequester for best DNA quality

Quantitation - how much DNA we have bc right amount of DNA is needed, if extraction was done correctly - to not waste time and money, and know species specific DNA

Profiling - w/ STR

Advantage of workflow

sensitivity - trace samples

Disadvantage workflow

Sensitivity - contamination, inhibitor

Solid phase extraction

Solid Phase Extraction: uses silica which binds DNA in high salinity acidic conditions and can be separated in basic conditions

Real time PCR

• It is done live, use fluorescent dye (probes, binds to sequence) as polymerase builds more copies , more dye will incorporate into them

• A dye binds to DNA (can bind to bacteria), all DNA

• A probe is more specific, binds to sequence chosen on DNA

Real time PCR difference

probe or dye, scanner special instrument to detect fluorescence, control of known concentrations

Why do real time PCR phases exist

because there is not enough nucelotides

Which sample has the most starting DNA in real time PCR

The first one bc it took less time to detect flourenscne 

Threshold

Threshold - set by own lab, follow NIST 

-Go to court w/ a match for inclusions if it passes interpreational threshold (sufficient DNA)

Electrophoresis

-separates molecules by size and charge 

Why at negative end in electrophoresis

bc DNA is neg. Charged - will move to the positive end cuz it repels

Why DNA at top in electrophoresis AND what does M stand for

DNA stuck at top, are larger and longer

M = molecular ladder

Disasvtanages of GEL electrophoresis

Messy, warp gel

Capillary electrophoresis advantages

A capillary is a thin tube

Avdntage: thin wall capillary, has high SA to volume ratio = heat is dissipated very quickly, so NO warping , low use of sample, done quicker

Capillary electrophoresis disadvantage

Very costly

Random Match Probability

:

• what are the chances two people could have that exact profile

Allele frequency

Allele freq = total # of times showed up / total # of ppl in database

Hardy Weinberg 5 conditions

1. LARGE POP

2. NO NATURAL SELECTION

3. NO MUTATION

4. NO IMMIGRATION

5. RANDOM MATING

Hardy Weinberg

Allele freq. In a pop. is going to remian constant if certain conditions are met (mutations, genetic drift, gene flow).

p^2+2pq+q^2=1

p^2 - AA          2pq - Aa             q^2 - aa

HARDY WEINBERG Can be used w STR profiles bc of  

1. Linkage - if i have a 7 at locus 1 it will not affect any other locus that were tested - independently inherited

2. Stable allele freq

Product rule

-Prob. that another given person could have deposited that sample

-the prob. Of independent events occurring together

(multiply everything in last column)     if last column not given,  use p^2+2pq+q^2=1

RAPD

-uses PCR

-dont know genome

-random amplification of polymorphic DNA w/ primers

-dont know target, but can still get a match between a known and an unknown w/ same random primers

RAPD Advnatages

Make up primers, dont need to study genome

-low cost

RAPD disadvantage

:

-Choose several different primers (trouble shoot)

-low resolution of gel

AFLP

-no markers to target, dont know genome

-both pcr and rflp based

Pcr adds sensitivity so doesnt need large amounts of dna 

1. DIGEST DNA w/ R.E. TO CREATE FRAGMENTS

2. LIGATE THOSE FRAGMENTS W ADAPTERS (MAN MADE CUZ WE USE WHERE THE R.E. CUT)

3. ADD PRIMERS to amplify

technique that allows us to very accurately profile an individuals DNA without having any knowledge of the genome

Works bc a restriction digest is done with restriction enzymes that will generate discriminatory patterns for any specific DNA , generate a visual match

AFLP advantage

Low amounts of DNA

More discriminatory than RAPD

AFLP disadvantage

-takes longer

-more coslty