Module 3

Why is it important not to handle the optical plate except by the edges, even when wearing gloves?

Because any smudges, dusts, fingerprints on the bottom of the optical plate can affect the fluorescence detected by the instrument

Why is it necessary to work under a fume hood when performing organic extraction?

Because the phenol/chloroform is a mutagen and is hazardous to our health when it is inhaled or in contact with our skin or eye

When the trace lab identifies sperm on a specimen, the sample is rated. What is this rating method and how does each sperm rating correspond to the number of sperm observed microscopically?

1+ 1-9 per slide 2+ at least 10 per slide 3+ 11-19 per field 4+ 20+ per field N/O- none observed

What are the minimum and maximum volumes of a Microcon filter?

10-500 ul

Explain electro kinetic injection. What factors may affect this type of injection.

A positive voltage is applied to the capillaries which gets immersed into the DNA solution and because DNA is negatively charged this pulls the DNA into the capillaries. Salt concentration and formamide can affect the amount of DNA that gets injected into the capillaries. Formamide maintains the conductivity. When salt [ ] increases, less DNA gets injected because it is competing with the salt.

Explain the difference between the analytical threshold and the stochastic threshold.

Analytical threshold- is a threshold where a peak can be reliably said to be a true allele and not baseline noise Stochastic threshold- a threshold used to say that a homozygous peak is indeed a homozygous peak and that no dropout has occurred

During sample normalization and amplification?

Bench area and equipment cleaned. DNA is added after master mix. Open one tube at a time, one pipettes tip per sample.

What steps are taken during DNA extraction to limit potential contamination?

Bench area, equipment, and instruments are cleaned with DNAaway or other decontaminating reagents. Pipette tips, scissors, and scapels are changed for each item. PPE worn, disposable bench paper are used. Questioned and known samples are procesed at diff time/space. Pre-extraction room is separate from post-amp room

What is capillary failure and why does this occur?

Capillary failure is when samples and ILS are not detected resulting in bad resolution or low RFUs. This occurs when DNA or other enzymes bind to the inner coat of capillaries

Write a short explanation of the three phases of DNA amplification.

Denature tin- hydrogen bonds are broken either due to heat or chemical and the 2 strands separate. Anneal- primers bind to the flanking region of the STRs Extension- Taq polymerase stats copying the sequence of interest

If a sample were over-saturated, what actions should be taken?

Dilute the sample, re-amp, re-run it, less injection time

How does the EZ-1 differ from other extraction procedures?

EZ-1 is an automatic procedure and is similar to DNA IQ in such a way that it also uses a magnetic resin and when chaotropic salt are present it binds to the silica paramagnetic beads and elites under more alkaline conditions

What is a primer?

Is a short oligonucleotide to help the DNA polymerase recognize a starting point for it to start extension of the copying process

What is microvariant?

It is a incomplete repeat sequence. It is usually one basepairs less in a tetranucleotide resulting in alleles such as 9.3

How does DNA IQ differ from other extraction methods?

It is a solid-phase extraction. It uses a silica magnetic resin. Lysis buffer lysis the membrane and DNA binds to the silica which will move to the side of the tube when a magnet stand is present, while other impurities are washed away. Under more alkaline conditions, the DNA will elute from the silica magnetic resin

Briefly describe how rt-PCR is helpful in determining the quantity of DNA present in a sample.

It records the amount of amplicon a cycle by cycle. Thermal cycling, fluorescent detection occurs at the same and thus provides accurate quantitation

How has the Taq Gold polymerase been modified?

It requires a pre-heating step for it to be activated. Helps prevent mispriming and primer/primer dimer annealing

What is a non-template nucleotide addition?

It's when Taq polymerase adds an addition nucleotide to the 3' end of the strand during the copying phase resulting in one basepairs more than the true allele

What is a negative control? When is it processed? What are the expected results from a negative control?

Negative control is processed during amplification to ensure that all the reagents used in amplification is free of DNA and therefore result should be no DNA. It contains all the amp reagents minus any DNA

When the internal lane standard is not usable, can the results be accurately interpreted?

No, because the samples are sized based on the ILS

How many standards can be deleted from each standard set for the data to still be acceptable?

One concentration from each standard can be removed (2 data points) or the 2 lowest concentration (G1, G1) 0.0032ng

Plexor HY uses a quencher, dabcyl, as part of the rt-PCR reaction mix. Briefly describe how the quencher functions in determining DNA quantity

Plexor HY measures the amount of DNA by decreasing in fluorescence. The dabcyl quenches the fluorescence that is attached to the primer

What is a positive control? When is it used?

Positive control is known to contain DNA and is process during amplification to make sure the amp was successful

What is preferential amplification?

Preferential amplification is when an allele is amplified at a higher rate, which result in heterozygote imbalance or allele dropout in a heterozygous loci. Usually STRs with shorter base pairs amplify more efficiently than larger STRs

What is the general procedure for a differential extraction?

ProK and SDS is added to break down membrane of e-cells. Tube is centrifuged and the supernatant is removed to a new tube which contains the e-cell fraction and the pellet which contains the sperm-cell fraction is then listed with Prok, SDS and DTT to break the sperm nuclear membrane

What is a reagent blank? When is it processed? What are the expected results from a reagent blank?

Reagent blank is processed during extraction and it contains all the reagents that were used in a particular extraction method minus extracted DNA. Expected results should be no DNA

How do negative controls and reagents blanks differ?

Reagent blank is processed during extraction to monitor no contamination occur throughout the whole DNA typing processing and negative control is process during amplification to make sure all amp reagents are clear of DNA

What is the purpose of spatial calibration and when should this be performed?

Spatial calibration creates a relationship when a signal is emitted by the capillary and the position where the signal actually gets detected by the CCD camera. Should be performed when arrays get changed, instrument been moved, laser door opens, laser camera been moved

What is the purpose of a spectral calibration and when should this be performed?

Spectral calibration- creates a matrix to correct for the overlapping fluorescence spectra of the dyes. Should be performed when new spectral dyes are used, capillary arrays are changed/removed, laser door open, instrument moved

What is a stutter peak? How is a stutter peak differentiated from a true allele?

Stutter peak is one repeat more or less than a truer allele and is a result of strand slippage during PCR. When a sample is known to be from a single source, usually a stutter falls within the stutter threshold and if it is above the threshold the sample might be overloaded when the sample is a mixture and the stutter peak is above the threshold, it would usually be called a true allele

Give a brief explanation of fluorescence detection.

The photon of a laser excites the fluorescence dyes in the primers. The fluorescence goes from a ground state to an excited state and when the fluorescence is going back to a ground state the light that is emitted has a higher wavelength and gets recorded by the detector.

What is the function of DTT in a differential extraction?

To break the disulfide bonds in sperm nuclear membrane

Why is proteinase K used in extractions?

To degrade the proteins and enzymes in the cell such as nuclease

Why is an internal positive control (IPC) included with each sample that is quantitated?

To ensure that the reagents, assay and the instrument is working properly

Why is a standard curve generated?

To help determine the concentration of the unknown samples. The Ct values are given a point on the standard curve to determine the concentration

A pre-extraction wash step is recommended when analyzing hair? Why is this?

To remove any contaminants and impurities such as dyes, hairs prays, gel

Why is it necessary to print primer peaks for negative controls, reagent blanks and negative samples?

To show that primers were indeed added to the samples and not just running blanks

Describe the three validated EZ1 purification protocols

Trace- can be used on all samples Tip dance- the filter tip moves back and forth and are good for blood cards and buccal swabs bc the moving of the tip will not clog the tips. Good for solid material. Large volume- can be used for volumes up to 500 up. Efficient for purification of dilute samples with low DNA concentration

If in GeneMapper, all size quality symbols are red stop signs, name two things that could be done to troubleshoot this problem.

When it is usually all red stop sign symbols this means that the ILS range is not wide enough. To trouble shoot-> change the starting and ending ILS points or re-run the samples.