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In DNA extraction protocols, what is the purpose of proteinase-K?
Break peptide bonds in proteins to produce small peptides.
Which best describes the nucleotide content within different cell-types of the human body?
The DNA content is the same and mRNAs have unique patterns of expression.
EDTA is an example of a _____ ______.
Chelating agent.
CHELEX extractions provide _____.
ssDNA.
OH NO! You forgot to perform the spin steps using Buffer AW1 and AW2, meaning you went straight from the spin after adding ethanol (Step 6 in the lab protocol) to spinning with Buffer AE (Step 9 in the lab protocol). Where is the DNA after the centrifugation step 6?
Bound to the silica membrane.
OH NO! You accidentally used a p20 pipette instead of a p200 during your elution step with buffer AE. As a result of this error, the DNA would be eluted from the column in _____ uL, instead of the specified _____ uL.
10, 100.
OH NO! You accidentally used a p20 pipette instead of a p200 during your elution step with Buffer AE. If you caught this error immediately, how could you correct it?
Perform a second elution step with the remaining volume of AE to elute the residual DNA from the column.
The passive reference dye used in the Quantifiler Duo DNA quantification assay is called _____.
ROX.
The Quantifiler Duo _____ mix contains light sensitive probes.
Primer.
The process of diluting or concentrating your DNA sample to an optimal range for the multiplex PCR is called _____.
Normalization.
At what stage of the qPCR do probes fluoresce?
Extension.
What is your interpretation of the following Quantifiler Duo results?
Target | Cт | Quantity |
(ng/µl) | ||
Human | Undetermined |
|
IPC | 28.45841776 |
|
Male | Undetermined |
|
No human DNA was detected.
| ||||||||||||||
A mixture of male and female DNA is present.
OH NO! You used a pipette without a tip! What would be the most likely outcome of this blunder?
Liquid would enter the pipette body and contaminate future transfers.
Why is it important to ensure that pipettes are held upright while there is liquid in the tip?
To avoid liquid getting into the pipette body and contaminating future transfers.
OH NO! You dispensed a liquid from the measuring stop instead of the blow out stop. What would be the most likely outcome of this blunder?
You would dispense a smaller volume than intended.
You perform a single-locus RFLP on locus D1S7 and after developing the Southern blot, you notice there is only one-band in the lane corresponding to suspect 1. The most likely explanation(s) for this result is:
suspect 1 is homozygous for locus D1S7.
The CODIS marker D7S820 is found within:
A noncoding region on chromosome 7.
The following treatments denature DNA from its double-stranded form:
Low ionic strength salts
Urea
Formamide
High temperatures (>95 Celsius)
What is the main advantage of using quantitative PCR for human DNA quantification?
It is designed to quantify the total amount of amplifiable human DNA and human male DNA simultaneously.
What is one limitation of using quantitative PCR for human DNA quantification?
In highly degraded samples, assays that detect short target sequences will amplify more product than those that detect long sequences due to the breaks in the longer DNA fragments.
Why are passive dyes used in quantitative PCR assays and list a specific technical aspect of the assay they correct.
They are used to correct and normalize the minor differences between fluorescent signal detections across wells in the same plate. When a plate is centered under the light filter or detector, the wells in the center of the plate will have a shorter light path than those on the perimeter of the plate, resulting in slight differences in the signals recorded. The passive reference dyes are able to adjust these signal recordings so that the output of data is even across all wells in the plate.
What is cycle threshold and how is it used in qPCR to quantitate unknown samples?
The cycle threshold (Ct) is the number of qPCR cycles required for the fluorescent signal to exceed baseline noise and become detectable. We can compare unknown Ct values to a standard curve of known concentration samples in order to quantitate the qPCR sample of interest.
A mixture of primers that have sequence substitution of different bases but amplify the same locus are called:
Degenerate primers.
The goal of multicomponent analysis is to:
Correct for spectral overlap.
How are PCR products fluorescently labelled using the Identifiler Kit?
Using primers labelled with fluorescent dyes.
Following the Identifiler PCR amplicons are diluted in deionized formamide and injected into which instrument?
Genetic analyzer.
What is the purpose of 9947A in the Indentifiler assay?
It is used in a PCR positive control reaction (Human DNA correctly detected).
Heating the PCR products to 95 degrees Celsius for several minutes followed by rapid cooling to ~4 degrees is commonly referred to as:
Snap-cooling.
The acronym CODIS stands for the:
Combined DNA Index System
One of the reagents you will handling to set up your PCR is the AmpFSTR PCR Reaction mix. List two components that are likely part of this mixture.
MgCl2 and bovine serum albumin in buffer.
List two reasons why formamide is added to PCR products prior to the genotyping run?
It denatures double-stranded DNA and dilutes salts within the sample. It also prevents complementary ssDNA molecules from interacting with each other and re-annealing.
Describe two problems that occur when too much DNA is added to the PCR?
Incomplete adenylation due to the excess of DNA templates relative to the amount of polymerase molecules in the reaction. Off-scale data such as pull-up caused by the fluorescence intensity from the PCR products exceed the linear dynamic range for detection by the instrument.
What is the main advantage of using hot-start Taq polymerase for forensic applications?
Hot-start Taq polymerase is a modified Taq polymerase which only activates after a heat shock of 95 degrees celsius. The main advantage of hot-start Taq polymerase is that it decreases or eliminates the chances of non-specific product formation in the sample, as it cannot become activated at room temperature or during reaction setup.
What is the main difference between hot-start Taq polymerase and hot-start PCR?
Hot-start PCR involves adding unmodified taq polymerase into the reaction at a higher temperature than normal to minimize mis-priming, while Hot-start Taq polymerase refers to a modified Taq polymerase which only becomes activated at elevated temperatures.
List two reasons why forensic labs are hesitant to perform PCR with >28 cycles?
PCR with >28 cycles can produce off-scale data, which is observed for several allele peaks at 31 cycles. This can raise the baseline of detection and create off-scale peaks which both mask and alter the presence of alleles and the height of their peaks. Secondly, using more cycles can increase the chance of amplifying unwanted products which may obscure true alleles. The Amplifiler Kit, commonly used in forensic analysis, works optimally with 28 cycles when the recommended conditions are met, amplifying 1.0 ng of DNA reliably.E
What standard is used to convert the raw output of electropherogram migration times into peak sizes?
Internal size standard.
How are allele frequency tables calculated?
They are determined by counting the number of times each allele is observed in a population sample, and dividing by the total number of alleles observed.
List two factors to be considered when constructing and using allele frequency tables.
Relatedness of individuals in the sample and linkage equilibrium.
Define random match probability (RMP).
It is the estimated frequency of a specific profile occurring in a specific population based on the observed allele frequencies for that population.
Which agency/lab cannot add DNA profiles generated from crime scene samples to the National DNA Data Bank?
The Federal Bureau of Investigation Lab.
List two characteristics that make the HV1 and HV2 markers useful when working with a degraded sample:
The circular structure of mtDNA and the high copy number of mtDNA relative to nuDNA.
The presence of more than one mtDNA type within a single individual is known as:
Heteroplasmy.
Mitochondrial DNA and Y-STR markers are often used to identify parental lineages and can both be used to produce:
Haplotypes.
