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Forensics

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Polymorphism
Existence of 2+ variants at significant frequencies in population
ex. blood group antigens, eye color, hair color, biochemistry polymorphisms, DNA polymorphism
used in paternity testing and criminal cases
2 Critical Discoveries
1. Discovery of potential polymorphisms
2. PCR
Sequence Polymorphism
Sequence is the same except for a few bases, recognized by restriction Ez
Length Polymorphism
Areas of DNA with small bases repeats
ex. 4 bases repeated: 1 chromosome has 3 repeats (12 bases) and the other chromosome has 2 repeats (8 bases)
STR's
STR's
Repeats of 4-5 bases, primer bind site for forward (A) and reverse reaction (B) - A and B flank a single copy of DNA
Allele for genetic loci
Version or variant of a gene or DNA sequence
Allele for STR loci
Designated number of repeats
STR loci characteristics
1. Inherited in a Mendelian fashion
2. Found in non-coding or non-transcribed regions
3. Found on all autosomes and X and Y chromosomes - STR loci on Y are male specific
4. Used for forensic testing, repeats of 4-5 bp
5. Panels used for forensic ID and paternity currently test for 13, 15 or currently 20-24 loci in a panel which is way more than necessary
Types of Biological Evidence
All nucleated blood cells
Semen
Epithelial cells: saliva, vaginal cells, skin cells, buccal cells
Tissues, bones, teeth, hair, fingernails and scrapings
Where is evidence found?
Clothing, bedding (stains and wear areas), things people use (glasses, cigarettes etc.), weapons, vehicles, bullets, cartridge casings, surfaces
Prior to testing
-Chain of custody- document every step in the lab and all personal involved in each step
-Analysts locate areas to be tested and may identify type pf body fluid present: specific color tests and microscopy to identify
Presumptive tests
Indicated but not conclusively prove the presence of body fluids like blood, saliva, semen
Confirmatory tests
Confirm presence of fluid, color tests confirm fluid, microscopic identification of sperm
Process of ID stain
-Data Collection: Sample deposited--> sample collected--> DNA extraction --> quantitation --> PCR amplification --> CE separation/detection
-Data Interpretation: CE (sep/detection) --> signal --> peak --> allele --> genotype --> profile
-Compare profile to other evidence
Lab organization to prevent contamination
Separate areas in lab for each step, one-way flow of work
DNA Extraction Choice of Method
Could sample be degraded? Use what know of sample to determine this
How much sample is available? Is there enough sample for each test or is it sexual assault sample (dealt with differently)
DNA Extraction Steps
1. Cell Lysis: use detergent (SDS) and a protease (Proteinase K) and buffer, to chew up proteins around DNA so DNA can more easily go into solution
2. DNA Purification: many methods
Methods for DNA Purification
-Phenol Chloroform (P/C)- not so much anymore
-Binding of DNA to silica- in magnetic field, ex. Qiagen products
Direct Cell Lysis Procedures
Lysis of Cell and nucleus occurs and releases of DNA into a PCR compatible solution- no further DNA purification
Phenol Chloroform Extraction
-dsDNA more soluble in water than the proteins other cellular components
- when extracted in presence of phenol- DNA remains in aqueous layer and proteins and other cellular components sit at the phenol and water interface: hydrophilic and hydrophobic parts of molecules are more stable there
-organic phase on bottom and aqueous layer on top
Silica Based Extraction
Sample + salt + SDS + PK, incubate cells to lyse cells--> cells lyse--> DNA binds to silica: proteins and contaminants pass through--> wash with ethanol--> add elution, H2O and DNA released back into solution
Sexual Assault Sample
-Microscopy Sample: pale red= nuclei and small bright red= sperm
- use differential extraction on vaginal swab to separate the two
Differential Extraction
-used in sexual assault vases
- remove portion of the mixed stain: have sperm and epithelial cells mixed--> add SDS, EDTA, PK, cell lysis buffer --> incubate and centrifuge --> sperm pellet out--> remove supernate --> female fraction
- After sperm pellet--> add SDS, EDTA, PK, and DTT which lyses sperm heads--> male fraction
PCR general
- in vitro chemical reaction, allows specific DNA or RNA seq to be coped
- copies produced at an exponential rate when chemistry in optimal
-components: DNA template, dNTPs, ssDNA primers, thermal stable DNa pol (Taq), MgCl2 to sustain Ez activity, buffer
PCR First Cycle
Denature strand--> primers bind to specific sequences--> primer sequence determines direction of extension --> have another primer on other side of sequence --> nothing to stop
PCR Second Cycle
-Denature again--> primers bind at original template, DNA is the same every time but primers also bind to template strand--> extend
-1st amplicon~ specific length copy from long copy
-original template doesn't leave strand
- exponential doubling
Separation of PCR products requirements
-Chemistry: STR loci amplified with fluorescent dye labeled primers, DNA denatured prior to electrophoresis, electrophoretic separation done using acrylamide type polymer in thin glass capillary
-Hardware: CCD camera, laser, electrodes, pump block, auto-sampler
-Software: data collection, color separation, peak sizing, allele designation
Separation of PCR products process
-Primer on forward strand has fluorescent flag: only on forward not reverse b/c the sequence is complementary
-Sample tray and DNA migrates through 36cm capillary not coated at one point--> laser directed to this and every time the dye passes the window the laser excites dye--> dye emits light thats picked up by CCD camera
DNA profile
-Colored profile in different colors
-3 Pieces of information: 1. allele size, number of repeats (most important) 2. height of peak: the more DNA the higher the peak 3. calc of size of peak in base pairs
Allele/ locus drop out
- a limit to DNA profile testing
- as amount of DNA becomes smaller, lose information
- can lose one allele or a heterozygous pair OR loss of both alleles
More than one person in a sample
- if didn't know genotypes, just had mixed profile- wouldn't know difference b/w 2 people
- when ratio of 2 DNA samples in profile are different can get more information
- 2 contributors: 14 combos of how mix up 2 people
- 3 contributors: 150 combos of mixing up people
Challenges of Mixed Interpretation
-Difficult to determine the number of contributors with certainty, can only determine the minimum number of contributors
-Difficult to determine ratio of contributors with certainty
-Difficult to determine whether any information has dropped below level of detection when amount of template DNA is low
National Database System
-National level: NDIA= FBI lab
-State level: SDIS= each state has one
-Local level: LDIS= different numbers in each state
Data Base Searches
-Convicted Offender DNA Profiles data base
-Case Evidence DNA Profiles data base
-New suspect DNA profiles and new evidence DNA profiles go into both and can be cross-referenced
Other DNA tests
-mitochondrial DNA
-differences at STR loci on Y chromosome
-parents of single nucleotide polymorphisms
-individual loci that code for or influence hair color, eye color and skin color
-loci that contain information about ethnic background
Haplotype
-genetic variants that are contiguous in a length of DNA and are inherited together without recombination: genomic segments inherited as discrete units
-mitochondrial DNA inherited as a haplotype from mother to child (boy or girl)
-most of Y chromosomes inherited as haplotype form father to son
Mitochondrial DNA testing
Extract and compare sequences
Y chromosome testing
-no recombination across 95% of chromosome
-loci associated as a haplotype
-inherited as a unit from father to son: can't tell difference b/w father, son, grandfather
-contains STR loci as well as single nucleotide polymorphisms
-specific Y STR loci can be amplified like other STR loci using commercially available kits
Sperm Fraction
-matched victim (female) but has a lot of sperm
-test with Y STR's
-mutation on area of Y that should have produced peak- not real common but common enough