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Pile Management Card
Forensics

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DNA Fragment Analysis
The primers used for PCR have dyes attached to them:
Blue, Green, Yellow (shown in Black), and Red
A camera in the instrument detects the different wavelengths of light being emitted by the fluorescent dyes as they move through each
individual capillary
Target DNA Locations
Short Tandem Repeats (STRs)
-STRs are specific sequences of DNA that are repeated a certain number of times
-We look at 15 different STR locations (loci) along with a gender determination marker on the X and Y chromosomes
amelogenin
gender determination marker
Identifiler STR Multiplex Kit
15 different STR loci and a gender
determination marker (amelogenin)

Fluorescently labeled primer sets (16 pair)
PCR in a Thermal Cycler
Specific locations of DNA are copied and then analyzed
PCR Efficiencies
The number of DNA molecules/fragments increases 2-fold with each cycle
Xn = Xm(1 + Ex)^(n-m)

Xn = # of molecules at cycle n
Xm = # of molecules at cycle m
Ex = efficiency of amplification; 0-1
n-m = # of cycles elapsed between n and m
Step 3. Three Phases of PCR Amplification
1. Geometric or Lag Phase
2. Exponential or Linear Phase
3. Stationary Phase
AB 7500
instrument is used to determine the amount of DNA present in a sample of extracted DNA using real time PCR
Measuring the quantity of DNA
The AB 7500 instrument is used to
determine the amount of DNA present in a sample of extracted DNA using real time PCR
It may be important to measure the amount of DNA … as there is an ideal amount of DNA needed for the next step (0.5-2 ngs)
Real Time PCR Assays (Quantifiler)
Most accurate and human specific
Y-specific system is also available (Quantifiler-Y)
Hybridization and End Point PCR Assays
Relatively accurate but time consuming
Human specific
Agarose Gel
Fast, but crude and relatively inaccurate method; OK for high quality samples
Step 2. DNA Quantification
Agarose Gel
Hybridization and End Point PCR Assays
Real Time PCR Assays (Quantifiler)
Measuring the quantity of DNA
Step 1. DNA Extraction
SIMPLE
Chelex, FTA
MORE CHALLENGING
Organic, Salting Out
COMPLEX
Differential, Bone/Teeth, Hair Roots
Steps for DNA Analysis
DNA extraction
DNA quantification
Amplification
Electrophoresis
Software analysis
Statistical analysis
Sample Types for Forensic DNA Analysis
Any human biological fluid or specimen
Non-human specimens (dog, cat etc)
Botanical specimens (seeds, leaves etc)
Microbial specimen (anthrax etc)
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