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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 |
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| 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 |
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| amelogenin |
gender determination marker |
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| Identifiler STR Multiplex Kit |
15 different STR loci and a gender
determination marker (amelogenin)
Fluorescently labeled primer sets (16 pair) |
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| PCR in a Thermal Cycler |
Specific locations of DNA are copied and then analyzed |
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| 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 |
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| Step 3. Three Phases of PCR Amplification |
1. Geometric or Lag Phase
2. Exponential or Linear Phase
3. Stationary Phase |
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| AB 7500 |
instrument is used to determine the amount of DNA present in a sample of extracted DNA using real time PCR |
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| 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) |
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| Real Time PCR Assays (Quantifiler) |
Most accurate and human specific
Y-specific system is also available (Quantifiler-Y) |
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| Hybridization and End Point PCR Assays |
Relatively accurate but time consuming
Human specific |
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| Agarose Gel |
Fast, but crude and relatively inaccurate method; OK for high quality samples |
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| Step 2. DNA Quantification |
Agarose Gel
Hybridization and End Point PCR Assays
Real Time PCR Assays (Quantifiler)
Measuring the quantity of DNA |
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| Step 1. DNA Extraction |
SIMPLE
Chelex, FTA
MORE CHALLENGING
Organic, Salting Out
COMPLEX
Differential, Bone/Teeth, Hair Roots |
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| Steps for DNA Analysis |
DNA extraction
DNA quantification
Amplification
Electrophoresis
Software analysis
Statistical analysis |
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| 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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