Extraction
What are the two basic steps of an extraction technique? And describe each.
Extraction - removal of biological material from substrate
Isolation - Break open cells. Separate and purify DNA
Why do we quantitate?
It is required by standards that the laboratory follows (QAS).
Helps optimize downstream testing.
Quality Check
What does PCR stand for and who developed this method?
Polymerase Chain Reaction invented by Kary Mullis
What charge is DNA and why? And in an electrical field, where will DNA migrate?
Negative - Phosphate Backbone
To anode, away from cathode.
What is a stochastic threshold?
What does PCIA stand for and what does each component do?
Phenol - Extracts proteins and polysaccharides
Chloroform - Separates lipids and fats
Isoamyl Alcohol - Prevents foaming to help visualize layers
What quantitation method do we use? What does this mean?
Real-time PCR
Allows us to monitor the PCR process in real-time
What are the three main steps of PCR? What occurs during each step?
Denaturation - two strands of DNA are separated
Annealing - primers bind to complimentary sequence in the sample DNA
Extension/Elongation - Optimal temp for Taq - will replicate the DNA strand
Electrokinetic injection - charged species will enter capillary and will move toward anode
What does RMP stand for? And what does this tell you about a profile?
Random Match Probability
The rarity of a profile in a given population.
What is added to a differential extraction that is unique compared to a regular organic extraction and what does this reagent target?
DTT - dithiotheritol
Disulfide bonds on the sperm head
What is the CT value? What does it mean if the CT value is high?
What is inhibition? What are two ways that an inhibitor can affect amplification?
PCR efficiency decreases due to the presence of an inhibitor, so sample may contain sufficient DNA but not produce a good quality DNA profile.
Inhibitor may bind to the DNA or bind to Taq.
What is POP-4? How does it work?
Performance Optimized Polymer
-Coat Capillary walls, contains pores which DNA will travel through and interact with. Longer pieces of DNA will travel slower because of more interaction.
What is a likelihood ratio? How is an LR typically set up for forensics statistics?
Ratio of two different hypothesis. LR will give you how many more time likely is one hypothesis compared to the other.
Prosecution Hypothesis
Defense Hypothesis
What binds to the beads in a chelex extraction? Why do you want these components removed?
Polyvalent metal ions (Mg). Reduce inhibition and degradation - MG is a cofactor for DNases
What is the standard curve?
What does the R2 value, Y-Intercept, and slope tell you about the curve?
Made up of samples of known DNA, should be linear.
R2 value - Precision of the standards
Y-Intercept - Accuracy
Slope - Efficiency
What is included in amplification reaction mix and what does each component do?
Buffer - keeps reaction at optimal pH
Deoxynuceloside triphosphates - building blocks for synthesis
Primers - flank region to be copied
Taq Polyermase - adds dNTPs to DNA template
MgCl & KCl - cofactors for Taq
BSA - inhibits inhibitors, minimizes their activity
What causes pull-up? How is pull-up identified?
Failure of spectral overlap - there are overlaps in the spectral of each dye that is identifiable. This is subtracted from a signal.
Directly underneath the real peak.
How do you determine a major contributor from a minor contributor in a DNA mixture?
You compare peak height ratios. Through validations we have guidelines that we will use to determine if two peaks could belong to the same person.
What is the name of the bead resin used in a Chelex Extraction?
Styrene Divinylbenzine Copolymer
What is hot start PCR and why do we perform this?
The taq is attached to an enzyme that inhibits activity until a certain temperature is reached.
Taq will start working on the sample before thermal cycling and will create non-specific binding products
How is an allele in a sample called in GeneMapper? Think ILS and Ladder
ILS contains fragments of DNA at a known length (BP) which is plotted linearly based size and data point (time). When a peak is generated it compares the time to the ILS plot to determine base pair size. This occurs also in your allelic ladder for every allele. Once you have a base pair size of your sample peak you compare it to the allelic ladder and which allele is the same base pair size. The number of that allele is then assigned to your sample peak.
How do you determine the allelic frequencies used for statistics?
Published by manufacturers. A population of individuals are gathered and their DNA profiles obtained. Count # of times you see allele in the population.