Forensic DNA Analysis (Butler)
Name two common PCR inhibitors in forensic cases. How do these inhibit PCR?
1) hemoglobin, 2) indigo denim dye. Bind to the active site of Taq Polymerase and prevent function
Which end of the DNA sequence of interest do PCR primers anneal to?
3'
How much DNA (mass) is enclosed in each cell?
6 pg (10^-12 g)
What are the advantages of STR typing over VNTR typing?
1) A narrow allele size range that permits multiplexing; 2) A narrow allele size range that reduces allelic dropout from preferential amplification of smaller alleles; 3) The capability of generating small PCR product sizes that benefit the recovery of information from degraded DNA specimens; and 4) Reduced stutter product formation compared to dinucleotide repeats that benefit the interpretation of sample mixtures.
What are the 5 components of a typical STR kit?
1) PCR primer mix, 2) PCR buffer (DTPs, MgCL2, etc), 3) DNA polymerase, 4) allelic ladder, 5) positive control DNA
What are three disadvantages of PCR?
1) The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA. 2) Amplification may fail due to sequence changes in the primer-binding region of the genomic DNA template. 3) Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without careful laboratory technique and validated protocols.
Name some solutions to overcome PCR inhibition
1) dilute the sample (thus diluting inhibitor as well) and re-amplify, 2) add additional DNA polymerase to overcome inhibitor, 3) additives (like BSA) 4) separation step (such as filtration) before PCR to separate inhibitor and DNA
What are the three results of a Q-K comparison?
1) inclusion, 2) exclusion, 3) inconclusive
Describe the 6 characteristics of successful PCR primer pairs.
1) must be specific to the target region, possess 2) similar annealing temperatures 3) not interact significantly with each other 4) must not interact with themselves to form "primer dimers" 5) must be structurally compatible. 6) the sequence region to which the primers bind must be fairly well conserved because if the sequence changes from one DNA template to the next then the primers will not bind appropriately.
Describe the types of controls used to monitor PCR.
1) negative control (PCR reaction mixture w/out DNA template, checks for DNA contamination) 2) positive control (shows whether or not any of the PCR components have failed or were not added during the reaction setup phase of experiments conducted)
How can one insure relative homogeneity between PCR samples?
By creating a "master mix" that can then be dispensed in equal quantities to each PCR tube
Name an advantage of blood as the sample matrix.
it is easy to see that a sample has been collected (as opposed to a colorless swab from a saliva sample).
What is multiplex PCR?
more than one region to be copied simultaneously by simply adding more than one primer set to the reaction mixture
What are the components of a PCR sample?
primers, dNTP, DNA polymerase
What is a PCR primer?
short DNA sequences that precede or "flank" the region to be copied. A primer acts to identify or "target" the portion of the DNA template to be copied. It is a chemically synthesized oligonucleotide that is added in a high concentration relative to the DNA template to drive the PCR sample. Considerable knowledge of the DNA sequence to be copied is required in order to select appropriate primer sequences.
What are desired characteristics of a preliminary test for biological fluids?
simple, inexpensive, easy to perform, non-destructive (does not impact processing of DNA later on)
Sometimes no DNA is detected using qPCR, yet a DNA profile is able to be drawn from the sample. Why?
stochastic variation can limit the reliability of qPCR results due to allele dropout. replicate testing is essential! Also, the PCR buffers between the qPCR and STR reactions may be different. If the STR amplification buffer contains a different polymerase or materials to enable overcoming PCR inhibition, then results may not be equivalent and the qPCR assay may not provide a true measure of STR typing performance
What factors impact the secondary transfer of DNA?
surface texture and sample moisture
What is a null allele? How can they be predicted?
the DNA template exists for a particular allele but fails to amplify during PCR due to primer hybridization problems. Potential null alleles resulting from allele dropout can be predicted by statistical analysis of the STR typing data. The observed number of homozygotes can be compared to the expected number of homozygotes based on Hardy-Weinberg equilibrium (Chakraborty et al. 1992). An abnormally high level of homozygotes would indicate the possible presence of null alleles.
Why is extracted DNA stored at cold temperatures (-20, -80 deg. celsius)?
to prevent nuclease activity
How can biological fluids be visualized at a crime scene?
using an alternate light source (ALS)
What characteristics are necessary for a multiplex PCR assay to work properly?
(1) the primer annealing temperatures should be similar and (2) excessive regions of complementarity between the primers should be avoided to prevent the formation of primer dimers that will cause the primers to bind to one another instead of the template DNA.
Name and describe the two purposes of PCR.
(1) to increase the number of molecules representing a specific target site, and (2) to attach a label, most often a fluorescent dye, that enables detection of the amplicons produced.
Presumptive tests for sperm test for which substances? Name some examples?
Acid phosphatase and prostate specific antigen (PSA). Fast Blue B.
Commerical STR typing kits work best with an input of DNA template around _________.
1 ng
Why is DNA extraction necessary prior to analysis?
Because proteins and other cellular materials surrounding DNA act as inhibitors of PCR
Describe the Chelex extraction method
Chelex is a chelating resin that binds Mg. Chelex denatures double-stranded DNA and yields single-stranded DNA from the extraction process. Thus, it can only be followed by PCR-based analyses. However, Chelex extraction is an advantage for PCR-based typing methods because it removes inhibitors of PCR and uses only a single tube for the DNA extraction, which reduces the potential for laboratory-induced contamination
What is differential extraction? What is this procedure used for?
Differential extraction is a modified version of the organic extraction method that separates epithelial and sperm cells. The differential extraction procedure involves preferentially breaking open the female epithelial cells with incubation in an SDS/proteinase K mixture. Sperm nuclei are subsequently lysed by treatment with an SDS/proteinase K/dithiothreitol (DTT) mixture. The DTT breaks down the protein disulfide bridges that make up sperm nuclear membranes. The assay works because sperm nuclei are impervious to digestion without DTT.
What is the purpose of adding EDTA to DNA samples? What does EDTA do?
EDTA prevents nuclease activity. Nucleases need Mg to work properly. EDTA binds up free Mg.
Describe DNA IQ method of extraction of DNA
First, the DNA molecules are reversibly bound to the magnetic beads in solution with a pH more acidic than pH 7.5 (the same as noted previously for the QIAGEN chemistry). A magnet is used to draw the silica-coated magnetic beads to the bottom or side of the tube leaving any impurities in solution. These solution impurities (proteins, cell debris, etc.) are easily removed by drawing the liquid off of the beads. The magnetic particles with DNA attached can be washed multiple times to more thoroughly clean the DNA. Finally, a defined amount of DNA can be released into solution via heating for a few minutes.
Is luminol destructive?
No. It has been shown to not inhibit DNA testing of STRs that may need to be performed on evidence recovered from a crime scene (Gross et al. 1999)
Describe QIAGEN Extraction of DNA
In this approach, nucleic acids selectively absorb to silica on a support, such as small glass beads, in the presence of high concentrations of chaotropic salts which disrupt hydrogen-bonding networks in liquid water and thereby make denatured proteins and nucleic acids more thermodynamically stable than their native folded or structured counterparts. If the solution is more acidic than pH 7.5, DNA adsorption to the silica is typically around 95% and unwanted impurities can be washed away. Under alkaline conditions and low salt concentrations, the DNA will efficiently elute from the silica material.
Describe the effects of too much and too little template DNA on the results of PCR amplification. What must be done prior to amplification in these cases?
PCR amplification of too much DNA results in overblown electropherograms that make interpretation of results more challenging and time-consuming to review. Too little DNA can result in loss of alleles due to stochastic amplification and failure to equally sample the STR alleles present in the sample. In these cases *normalization* is necessary. This involves diluting the sample down to the desired range or concentrating it by removing excess fluid.
What is low-temperature non-specific priming and how can it be avoided?
Regular DNA polymerases exhibit some activity below their optimal temperature, which for Taq polymerase is 72°C. Thus, primers can anneal non-specifically to the template DNA at room temperature when PCR reactions are being set-up and non-specific products may result. This can be avoided by initiating PCR at an elevated temperature, a process usually referred to as "hot-start" PCR. Hot-start PCR may be performed by introducing a critical reaction component, such as the polymerase, after the temperature of the sample has been raised above the desired annealing temperature (e.g., 60°C). This minimizes the possibility of mispriming and misextension events by not having the polymerase present during reaction setup. ***However, this is time-consuming when working with large numbers of samples, and opening sample tubes increases risk of contamination
Differentiate between simple repeats, compound repeats, and complex repeats
Simple repeats contain units of identical length and sequence, compound repeats comprise two or more adjacent simple repeats, and complex repeats may contain several repeat blocks of variable unit length as well as variable intervening sequences
Describe the 'Slot Blot' approach to DNA quantitation.
Slot blots involved the capture of genomic DNA on a nylon membrane followed by addition of a human-specific probe. Chemiluminescent or colorimetric signal intensities were then compared between a set of standards and the sample. ***the slot blot assay was not as sensitive as desired.
What are stutter products? How do they arise?
Stutter products are amplicons that are typically one or more repeat units less in size than the true allele and arise during PCR because of strand slippage (Walsh et al. 1996).
Differentiate between VNTRs and STRS
VNTRs are 8-100 bp in length while STRs are 2-7bp in length
What are the limits of negative PCR controls?
a single PCR tube (or other plasticware that the DNA samples pass through during sample processing) may contain contaminating DNA. If the contaminant DNA is present in only the negative control PCR tube, then the testing analyst may get a false impression that other samples in the batch are contaminated when in fact they are clean. If the contaminant DNA is present in one of the casework DNA samples, then a mixture may result between the contaminant DNA and the original casework sample—and the clean negative control would not be helpful in assessing that a contaminant is present in one of the tested samples.
Differentiate between "allele drop-out" and "locus drop-out"
allele drop out occurs when a single allele is lost, locus drop out occurs when both alleles are lost
Presumptive tests for saliva test for which enzyme?
amylase
What is stochastic fluctuation? How can it be avoided?
an unequal sampling of the two alleles present from a heterozygous individual. This results when only a few DNA molecules are used to initiate PCR. PCR amplifying the same DNA extract twice can result in different alleles being detected at a locus. This can be avoided by adjusting the cycle number of the PCR reaction such that approx. 20 or more copies of target DNA are required to yield a successful typing result. In addition, scientists can (1) stop testing or interpreting data before you go low enough to be in the stochastic realm, or (2) try to limit the impact of the stochastic variation by additional testing and careful interpretation guidelines based on validation studies
What is qPCR?
analyzes the cycle-to-cycle change in fluorescence signal resulting from amplification of a target sequence during PCR.
What methods do serology tests use?
antigen and serum antibody reactions
What is CT? (in real time PCR)
cycle threshold(CT) for calculations. The CT value is the point in terms of PCR amplification cycles when the level of fluorescence exceeds some arbitrary threshold, such as 0.2, that is set bythe real-time PCR software to be above the baseline noise observed in the early stages of PCR. The fewer cycles it takes to get to a detectable level of fluorescence (i.e., to cross thethreshold set by the software), the greater the initial number of DNA molecules put into the PCR reaction. Thus a plot of the log of DNA concentrations versus the CT value for each sample results in a linear relationship with a negative slope.
Under what circumstances may a result be deemed "inconclusive?
damaged DNA, too little DNA, complex mixtures
