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125 Cards in this Set
- Front
- Back
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What is an allelic ladder?
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-"fake" sample that contains the most common alleles that occur at a particular locus -DNA samples are compared to the allelic ladder in order to convert the base pair sizes into allele calls |
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What is a fluorophore?
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-molecule capable of fluorescence -a photon of excitation light is absorbed by an electron of a fluorescent particle, which raises the energy level of the electron to an excited state-->energy is emitted as a photon to relax the molecule back to ground state -fluorophores can be excited repeatedly -used as fluorescent probes |
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What is a primer peak? What does it look like?
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-any unassociated primers left over after amp -they "run fastest" due to being short sequences, and appear as "junk" before the LIZ size standard starts (at 75 bp) -"junk in the front" |
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What is the voltage applied to?
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-directly to sample
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What things can tell you if you have a mixture?
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-more than two alleles at a locus and at more than one locus -peak height imbalance in autosomal loci -peak height imbalance in Amelogenin |
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Which fluorescent dyes are used to detect the Globalfiler loci? The internal lane standard?
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-internal size standard= LIZ (orange) -D3, vWA, D16, CSF, TPOX= 6-FAM (blue)(shortest wavelength) -Yindel, Amelogenin, D8, D21, D18, DY= VIC (green) -D2S441, D19, Th01, FGA= NED (yellow) -D22, D5, D13, D7, SE33= TAZ (red) -D10, D1, D12, D2S1338= SID (purple) |
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What is a microvariant?
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-an allele that has incomplete repeat units
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What are the fluorescent dyes sensitive to?
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-pH -light -temperature |
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What are the problems associated with off scale (blown out data)? |
-stutter -pull-up - -A -increased baseline |
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Explain loci nomenclature... example: D8S1818, FGA... |
FGA: 3rd intron on alpha fibrinogen gene TH01: located in the 1st intron of gene for tyrosine hydroxylase CSF1PO: 6th intron of prot-oncogene c-fms |
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Where does the Argon laser come from? What wavelength does it produce light in? What are the recordings based on? |
-camera shocks the laser -readings based on: strength of fluorescence, time from when current applied to time camera captures fluorescence -488 and 814.5 nm **from injection to detection |
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What is LIZ? |
-internal size standard -compared to DNA sample in order to convert the raw data into base pair sizes -orange dye -added to each sample and ladder before put on CE |
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What is a stutter band? How is it differentiated from a true allele? |
-peak that's on repeat unit (4 bp) less than true allele -will be seen as a small peak before the main allele peak on egram -can be differentiated from true allele by dividing the height of the stutter peak by the height of the main allele, multiplied by 100 |
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Full name of our CE instrument? |
ABI 3500 Genetic Analyzer |
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Why does the polymer and buffer need to be at room temp before being put on the 3500? |
-as polymer warms, it will "de-gas" -adding cold polymer might introduce bubbles to system and affect resolution -buffer should also be used at room temp for same reasons |
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Describe the CE process... |
1. sample is injected 2. fragments move through capillary based on size 3. as fragments pass the detection window, laser excites the fluorescent dyes 4. each dye emits its max fluorescence at a different wavelength 5. emitted fluorescence is passed through a diffraction grating, fluorescent signals separated according to their wavelength onto a CCD camera 6. algorithms used to determine base pair size and allele calls (local southern/LIZ for bp, ladder for allele calls) |
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Define STR |
Short Tandem Repeats -multiple copies of a short identical DNA sequence arranged in direct succession in particular regions of chromosomes |
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Are all of our STR loci on different chromosomes? |
NO! -chromosome 5: CSF1PO & D5 (both on q-arm) **26.3 million bp apart** -chromosome 2: D2S441 & TPOX (both on p-arm) **66.7 million bp apart** D2S1338 (on q-arm) -Y chromosome: Yindel, Amelogenin, DYS391 -chromosome 12: vWA & D12S391 (both on p arm) **6.3 million bp apart** |
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How many peaks are in the LIZ size standard? |
-designed to size DNA fragment in the 20-600 bp range -provides 36 single stranded labeled fragments -each fragment is labeled with a proprietary fluorophore |
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5 desirable characteristics for STRs (the loci)? |
1. on separate chromosomal location (unlinked) 2. size range (between LIZ sizing std), fairly small 3. high discrimination power 4. low mutation rate (don't want issues with primer hybridization) 5. low stutter characteristics (longer repeat units to lower stutter) |
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What does POP-4 stand for? |
-performance optimized polymer -4= 4% linear (uncrossed-linked) dimethyl polyacrylamide |
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What is the resolution required in STR analysis? |
1 base pair |
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It is very difficult to achieve the +A form during amp. True or false? |
TRUE -otherwise you wouldn't need the final extension for 60 minutes at 60C |
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Why do we use a 6 dye system instead of 1? |
-allows for multiplexing -allows to amp more loci in on reaction -better separation |
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What is a dye blob? |
-artifact -occur when fluorescent dyes come off of their respective primers and migrate independently through the capillary--> possibly because fluorescent dye tags begin to break down over time -peaks are fairly broad and possess the spectrum of one of the dyes used for genotyping (usually in only 1 color) |
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What is a spike? |
-artifact -generally peaks of the same size and similar height (within an order of magnitude of each other) seen in all dye colors -typically seen in same position in all dyes -caused by: presence of air bubbles, urea crystals, voltage spikes, dust/lint on detection window, fluorescent material in either the formamide or polymer -appear as a very sharp peak in raw data -"voltage spike"= electrical surge |
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What kind of injection method do we use to introduce samples into the CE system? |
-electrokinetic: consists of transfer of negatively charged ions via an electromotive force -as current flows from the cathode (-) to the anode (+), the DNA sample is introduced at the cathode end of the capillary |
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Why is the Stokes Shift phenomenon important? |
-the difference (in wavelength) between positions of the band maxima of the absorption and emission spectra of the same electronic transition -because of the difference between the apex of the absorption and emission spectra, the shift permits the use of optical fillers to separate excitation light from emission light |
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Draw the Stokes Shift |
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What is an off-ladder (OL) allele? |
-an allele of a size that is not represented in the Globalfiler Alellic Ladder or one that does correspond to an allelic ladder allele, but whose size is just outside a window because of measurement error |
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Volt definition |
-unit of electric potential -measures how much pressure in an electrical current |
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What is a spatial calibration? |
-From SOP: maps the pixel positions of the signal from each capillary in the spatial dimension of the CCD camera -performed: if capillary is changed or move and if the instrument is moved -establishes a relationship between the signal emitted by each capillary and the position where that signal falls on and is detected by the CCD camera |
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Define electrophoresis |
-movement of charged molecules through a medium in an electric field |
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During data collection, the fluorescent signals are separated by a __(1)__ according to their __(2)__ onto a __(3)__ camera in a predictably spaced pattern. |
1. diffraction grating 2. wavelengths 3. charge coupled device |
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How long is the capillary? Inner diameter? |
-36 cm from injection to detection -50 um inner diameter |
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Explain sample stacking and why it's important.. |
-a method of stacking, or condensing DNA or other samples to improve electrophoretic resolution -results when samples are injected from a solution that has a lower ionic strength (formamide) than the buffer inside the capillary |
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What is pull-up? |
-artifact -observed as peak-beneath-a-peak, or as an elevation of the baselines for any color -caused by a "matrix failure" or the inability of the detection instrument to properly resolve the dye colors -usually a result of a peak of another color exceeding the linear range of detection for the instrument |
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Advantages of CE? |
1. performed in minutes 2. automated 3. samples contained within capillary (don't worry about contamination) 4. small sample amount needed 5. data readily available (easier to interpret than a slab gel) |
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Show how light is emitted from excited fluorophore.. |
1. photon from laser source excited the fluorophore from its ground energy state to an excited transition state 2. fluorophore undergoes conformational changes and interacts with its environment resulting in the relaxed singlet excitation state 3. a photon is emitted at a lower energy when the fluorophore falls back to its ground state |
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Why do we want single stranded DNA for CE, instead of double stranded? |
-single stranded pieces will be more uniform moving through capillary (v. double stranded which are bulkier) -need the copies of fragments of same size to come out at same time |
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What 3 sizes of STR loci and how they are related to stutter formation? |
1. shorter repeat units: mispair more often, and more stutter 2. longer repeat units: less likely to mispair, and decreased stutter 3. more complex: less likely to mispair because they need to pair more exactly, decreased stutter |
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Difference between a single copy gene and a double copy gene? multicopy? |
-single: occurs once in the whole genome and one pair of chromosome -multi:occur on more than 2 pairs of chromosomes -double: occur on two different pair of chromosomes |
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What happens during amp that is important for CE? |
-fluorescent tags on forward primers are added to DNA |
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Explain how a sequence variation in the PCR primer binding site could affect getting a DNA profile? |
-causes a null allele: DNA present, but not amplified -since primer specific, primers won't bind at all= no profile |
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What are RFUs and what do they correlate to? |
"relative fluorescent units" -correlates to the amount of DNA present -shown in peak height |
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Why do we use Hi-Di Formamide as opposed to non Hi-Di varieties? |
(Hi-Di = highly deionized) -used because formamide is highly deionized, so there are fewer ions in the formamide to compete with the DNA fragments for injection onto the capillary |
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What is a matrix and why do we use it? How is it created? |
-separates the 6 different dyes into distinct spectral components, making each dye run discrete -created during spectral calibration to determine amount of overlap between the different dyes -measured by running DNA fragments labeled with each of the dyes in separate CE injections, producing a matrix file -matrix used to subtract the contribution of other colors on overlapping spectra |
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What is the capillary made of? |
a hollow fused silica tube |
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In an egram, what are the X and Y axes? |
-Y: RFUs (relative fluorescent units) -X: time plotted as base pairs *time correlates to size of DNA fragment with smallest fragments detected 1st, and largest detected last |
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Describe Local Southern Method |
-uses the reciprocal relationship between fragment length and mobility -unknown fragment is surrounded by 2 known sized fragments above and 1 below...then 2 below and 1 above -results are averaged and size of allele determined |
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What is the accepted theory on the formation of a stutter band? |
-"strand slippage" -each molecule of TaqGold enzyme adds ~80 bases before it becomes inactive -the exhausted enzyme then detaches from the template and "breathing" occurs between the two complementary strands -an active enzyme then attaches to the template to continue the amp process and brings the 2 strands back together -sometimes the 2 strands do NOT align properly upon reattachment= slippage |
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What is the incorporation rate of DNA polymerase? |
2800 nucleotides per minute |
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What is a primer-dimer? How can it affect PCR results? |
-created when 2 primers bind to each other -will be preferentially amplified due to small size compared to the other PCR products -if 2 primers binds to each other, they won't bind to the sample= allelic dropout |
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What primers are dye labeled and why? |
-forward primers ARE dye labeled -reverse primers are NOT dye labeled because you would end up with split peaks during CE if both are labeled *forward and reverse DNA sequence are same length but different base sequence, which can affect migration through CE--> a different migration between the two primers would cause split peaks if both were labeled |
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Describe how the DNA polymerase works and its features and effects on the amplification process.. |
1. SNA heated/denatured, leave ssDNA 2. temp lowered, primers attach to DNA 3. DNA polymerase attaches to 5' end of ssDNA 4. annealing begins at beginning of primer sequence 5. polymerase then adds dNTPs until it reaches end of primer sequence |
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How long are primers typically? What happens if they're too long/too short? |
-between 18-30 bp -if too small: might not be specific enough -if too large: takes longer to hybridize without increasing specificity *larger amplicons are more likely to cross-hybridize with other primers and sequences in the reaction mixture--> can terminate DNA polymerization |
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What is the function of Tris-HCl in the PCR reaction mix? |
-maintains correct pH -temperature sensitive: as temp increases, the pH decreases |
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How many cycles are there in the PCR program used to amplify forensic samples? |
28 |
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Explain denaturation, annealing, and extension of DNA |
-28 cycles 1. denaturation: 94C, 10 seconds: denature dsDNA to ssDNA 2. annealing: 59C, 90 seconds: primers and polymerase attach to ssDNA 3. extension: 59C, 90 seconds: polymerase replicates new strand of DNA |
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Taq makes DNA products that have __(1)__ overhangs at their 3' ends. How does this affect DNA profiling results? |
1. adenine overhangs -polymerase puts an A residue at the end of each cycle -have final extension at 60C for 1 hour at end of 28 cycles to allow this to happen (ensures that samples are all in +A form) |
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What goes into the amplification reaction? |
-25 uL total -7.5 uL reaction mix -2.5 uL primer mix -15 uL of sample + TE buffer (depends on amp dilution calculations) |
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What is the general quantity of DNA that your sample can experience allelic drop-out? |
100 pg = 0.1 ng |
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Function of BSA in PCR reaction mix? |
-binds inhibitors -stabilizes enzymes during storage -prevents adhesion of enzyme to tube and tip surfaces: lets everything float in solution |
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What is the accepted theory on the formation of a stutter band? |
*not well understood -caused by the polymerase slipping off the strand during replication--> once polymerase has slipped off, there is a pause in replication and the parent and daughter strands are temporarily unpaired--> when the polymerase begins to move again, there is a mispairing of nucleotides |
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Can Taq polymerase be active at below 95C? How would this affect amp product? |
-YES, at/below 72C -because it's not TaqGold (which isn't active below 95C) -at room temp, primers can anneal non-specifically, resulting in non-specific products such as primer dimers -amps competing products and sample is amped less efficiently |
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Purpose of sodium azide in PCR reaction mix? |
-purpose of salt: assists with primer annealing -purpose of buffer: maintain pH between 6.8 and 8.3 |
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pH of TaqGold at room temp? at 95C? |
-room temp (22.2C)= 8.3 -95C= 6.9 |
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What DNA polymerase do we use in our amplifications? Why? |
-Taq polymerase: because human DNA polymerase denatures at high temps during a PCR run -specifically use AmpliTaqGold: which is inactive at room temperature, which prevents non-specific products from forming |
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What does MgCl2 do during PCR? |
-MgCl2: buffer included in PCR reaction mix -Taq needs Mg 2+ to function properly -too much Mg 2+ leads to base errors and excess of non-specific products -too little Mg 2+ leads to low yields of PCR products |
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What do the primers need? (primer design) |
-specific -similar annealing/melting temps -don't want them to bind to each other -binding site needs to be well conserved (not a site that will mutate as frequently or not as likely to mutate) |
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What does the annealing temperature depend on? What if it's too high/low? |
-length and composition of the primers: generally 5C below lowest melting temp of pair of primers used -annealing temp too low: primers will bind to anything (non-specific)= non-specific amplification -annealing temp too high: primers won't bind to anything and the likelihood of primer annealing is reduced |
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What are stochastic effects and how do we determine the level at which one could see these effects? |
-peak height imbalance or complete allele dropout -preferential amp of one allele over another -noticed when a small amount of DNA (degraded or LCN) is amped and causes allelic peaks to be unbalanced -determined by calculating heterozygosity: if less than 70%, then mixture or stochastic effects present -occur around 100 pg -"random effects" - prevent by adjusting PCR cycle number so degraded or LCN will be amped more |
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What reagent in the PCR reaction mix is temperature sensitive? Why is this important? |
-Tris-HCl -as temp is increased, pH will decrease -important for TaqGold, when pH falls below 7 the chemical moeities will fall off and TaqGold will be active |
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What is in the PCR reaction mix? |
-MgCl2: (Taq needs Mg 2+ to function -BSA: minimize inhibitors -Tris-HCl: ensures correct pH -dNTPs: in equal concentrations to minimize misincorporation errors (which would promote chain termination) -Sodium azide: preservative for reagents and assists with primer annealing |
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What is multiplexing? What is important about multiplex design? |
-amplifying multiple STR loci in one reaction -designed to minimize overlap: smallest allele at on locus is larger than the largest allele of the adjacent locus within the same dye -base pair range: a little more than 100 bp to a little less than 400 bp: need bp to be below 100 and above 400 to do the local southern method |
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How is TaqGold geneticall modified? |
-chemical modification in an amino group at a lysine residue that prevents the enzyme from functioning at a pH above 7 -at an initial incubation for 11 minutes at 95C, the Tris buffer lower the pH of the solution to below 7, which causes the chemical modification to fall off & TaqGold can work |
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What happens if you have too much Taq? Too little? |
-too much: extends non-specific products (amp non-specific products)= get an excess of non-specific products -too little: no PCR product= won't get good profiles, not enough to replicate enough of the samples |
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How do inhibitors prevent PCR from working? |
-interfere with cell lysis (during extraction) -degrade DNA -inhibit polymerase activity -common inhibitors (humic acid, sand, wood, dyes, leather) will CO-EXTRACT -problem because prevents getting a full/complete DNA profile |
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Solutions to overcome PCR inhibition? |
-dilute template DNA, which will dilute inhibitor, then re-amp -Add BSA to reaction to decrease effect of inhibitors (it will bind inhibitors) -add naOH for Taq inhibitors -separation techniques like microcon filters -add more Taq and lower DNA amount (can only have up to 25uL for reaction volume) |
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Preferential Amplification defintion |
-the unequal sampling of the two alleles present in a heterozygous locus, primarily due to stochastic (random) fluctuation arising when only a few DNA molecules are used to initiate the polymerase chain reaction |
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What impact does the use of AmpliTaq Gold have on the PCR process? |
-helps prevent high background and low specific product yield during a PCR reaction -inactive at room temp, prevents non-specific primer annealing from being extended -above problems occur when reaction set up at room temp |
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What is the full name of our thermal cycler? Are the lids heated, why? |
-ABI GeneAmp PCR System 9700 -have heated lids to prevent the PCR mix from condensing on the plate and tube lids |
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Why do we use 25 uL for amp reaction? |
-determined through validation studies -lower volume: sample may evaporate in thermal cycler -larger volume: would be harder/longer to reach thermal equilibrium and would need to increase cycle time |
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The amplified volume for the positive control should be what? |
Combine 10 μL of control DNA (0.1 ng/μL) with 5 μLof TE buffer... giving 1 ng of DNA |
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What does dNTPs stand for? |
deoxynucleotidetriphosphates |
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What is a primer? |
-short sequence of DNA that precedes the region of DNA to be copied -two primers are included for each region of DNA that needs to be copied, called the forward and reverse primers |
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What kinds of samples show stochastic effects? |
-mixtures -degraded -low template/low level |
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What is an oligonucleotide? |
short ssDNA molecule |
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What is DNA polymerase? |
-enzyme that functions to catalyze the formation of phosphodiester bonds between the nucleotides during DNA replication and synthesis -recognizes the primer sequence and adds nucleotides in the 5' to 3' direction |
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What does stochastic mean? |
"random"= can't reproduce |
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Explain the purpose of the 60 minute hold at 60C in the PCR process.. |
-used for letting the amp process finsih, and to allow the polymerase to add its 3' adenine residue -if this step isn't performed, then we would have strands with 1 nucleotide short and we would get an allele with a repeat unit short (getting "shoulder peaks" |
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Taq lacks this type of enzymatic activity? How does this relate to the concentration of dNTPs? |
-lacks 3' to 5' exonuclease function (meaning it can't go back or "proofread" mismatched bp) -means concentration of dNTPs has to be correct so that we don't have a lot of mismatched base pairs -concentration of dNTPs in equal between A, T,C,G |
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Do you want your primers to form hair pins? |
-No! because they won't be able to bind to DNA because it's binding with itself -forms when primer is self-complementary |
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Are the dNTPs in equal or unequal concentrations? Why? |
-in equal concentration to minimize the misincorporation errors -misincorporation promotes chain termination (chain termination restricts amp of defective molecules and helps to maintain fidelity) |
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What is the plateau effect and how does it affect your DNA sample? |
-final amp phase= depletion of substrates (less substrates like dNTPs, primers...)
-non-specific products (resulting from mispriming events) may begin to amp preferentially over the sample which leads to allele drop-out |
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What happens to your amp product if you have too much or too little DNA? |
-too much: promote binding of primers to non-specific sites (non-specific binding) and effects pH of reaction and throws things off balance -too little: preferential amplification |
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What is the recommended concentration of DNA to amp? |
- 0.125 – 1.25 ng per amplification (0.0125 to 0.125 ng/uL) -can go as low as 0.5 ng if sample exhibits inhibition |
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What is a non-template nucleotide addition? |
-replication of DNA can end in 2 ways: 1. Taq adds adenine at end of sequence before it falls off (+A) 2. falls off without adding the extra adenine (-A) *ways to favor +A: final extension at 60C or adjusting primer sequences |
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Is preferential amp a problem? What types of samples doe sit seem to occur in more frequently? |
-YES! it's a problem: can result in an incorrect or ambiguous genetic typing of the sample -occurs in samples with too little input DNA (inaccurate quant or no quant): inhibitors may be present |
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What is the function of a primer? |
-primers signal to the polymerase where to begin replication and where to stop replication -it's complementary to the target DNA strand (binds to the DNA sequence preceding the desired area to be amped, a well conserved area) |
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Where do primers come from? |
-natural DNA replication: made within cells usually short strand of RNA -forensic biology: manufactured, fluorescently labeled, included in commercial kit |
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What is the most important thing in PCR? |
PRIMER DESIGN |
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What is PCR? |
-Polymerase Chain Reaction -enzymatic process where a specific region of DNA is replicated over and over again to yield many copies of a particular sequence |
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Draw a simple schematic of the CE process.. |
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What is the acceptable peak height ratio for heterozygous loci in known reference samples? |
50% or greater |
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What does "off-scale" mean? |
-fluorescence intensity exceeds the linear dynamic range for detection by the instrument -can cause: high stutter, pull-up, and -A |
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What is a spectral calibration? When is it performed? |
-creation of a matrix file to determine the amount of spectral overlap between the 6 fluorescent dyes -performed when: maintenance performed, run conditions change, instrument conditions change (replaced laser or CCD camera), use new dye set, changed capillary length, problems like uneven baseline and pull-up |
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How to check sizing precision? |
-check the 250 bp peak of LIZ -values must be within +/- 0.5 bp |
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Factors that affect how well a fluorophore will emit light include? |
1. Molar extinction coefficient: the ability to absorb light 2. Quantum yield: efficiency of emitting light 3. Photostability: ability to repeatedly fluoresce 4. Dye environment: pH, temp, solvent, quenchers |
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What could you do to fix blown out data? |
-amp less of the sample (adding more TE) -dilute whole sample -shorter injection time |
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What two things affect how fast a DNA fragment moves through the capillary? |
-voltage applied -size of fragment |
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What wavelength does the argon-ion gas laser produce light? |
488 nm and 514.5 nm |
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How many peaks in the internal lane standard are needed for data analysis? What algorithm is used? |
-The Local Southern Method: is the common algorithm for determining DNA fragment size - two sizing peaks on each side are used in calculation -the 250 bp in LIZ isn't used in calculations (does not give reproducible sizing results) *Global southern can also be used: involves using all of the size std peaks and fitting them onto a best fit size calibration line |
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Advantages of having the STR range narrow? |
1. easier to amplify degraded samples 2. reduces allelic drop out 3. reduces PCR amp time 4. allows for multiplexing |
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Why do we use POP-4? |
meets following specifications: 1. detect alleles differing in size by a single base pair 2. size alleles of the same length with a precision of below 0.15 nucleotide std deviation 3. requires less analysis time per sample 4. provides capillary life of at least 100 injections 5. provide highly denaturing environment for DNA samples |
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How many loci does the Globalfiler kit amp? |
-21 autosomal STR loci -1 Y-STR -1 polymorphic marker on Y chromosome -1 sex determining marker *Total= 24 |
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What changes are there from Identifiler to Globalfiler kit? |
-addition of DYS391 and Y-Indel -TPOX reverse primer redesigned to optimize marker spacing -addition of 8 new SNP-specific primers for D3, vWA, D18, D19, Th01, FGA, D5, and SE33 loci |
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Explain non-nucleotide linkers.. |
-used in primer synthesis for D19, vWA, CSF, D2S441, Th01, FGA, and D12 -non-nucleotide linkers placed between the primers and the fluorescent dye -enable reproducible positioning of the alleles to facilitate inter-locus spacing (allows for efficient separation of all 24 markers) |
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What is the volume of reagent blank in an extraction batch? |
-should be the largest volume added for other samples in the batch, or 15 uL |
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How many capillaries does the 3500 use? |
8 capillaries |
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What does the 3500 do during a run? |
1.prepares capillary- pumping fresh polymer solution under high pressure from the polymer delivery pump to waste position in cathode buffer container (CBC) 2. electrokinetically injects sample into capillary using low-voltage 3. washes capillary tips in rinse position of CBC, then returns capillary to buffer position of CBC 4. ramps voltage up to constant voltage, creating high electric field that pulls negatively charged DNA through separation polymer 5. in detection cell, the dyes attached to DNA are excited by narrow beam of laser light (which is emitted as a longer wavelength light) 6. 3500 captures fluorescent light and converts lights to multi-dye data for entire run |
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How often should the water trap/pump trap be flushed? |
once per month, with nuclease free water |
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Why do we do a spatial calibration on the 3500? |
-3500 data collection software uses images collected during the spatial calibration to establish a relationship between the signal emitted by each capillary and the position where that signal falls on and is detected by the CCD camera -should be performed after a capillary array is installed,when detector door is opened, when detector cell is moved, after the instrument is moved |
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During a spatial calibration, what does the software calculate? |
-average peak height= (sum of all peaks/number of peaks) **8 capillary threshold is 6400 RFU -uniformity (or peak height similarity)= (standard deviation/average peak height) **0.2 threshold -capillary spacing= (max spacing-min spacing) ** 2 pixels threshold |
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Why do we do a spectral calibration? |
-it creates a de-convoluted matrix that compensates for dye overlap (reduces raw data from the instrument) in the 6 dye data stored in each sample file -should be performed when a dye set is being used that has not been previously calibrated, when a capillary array is changed, when service has been done, when decrease in spectral separation (pull-up/pulldown in peaks) is observed in raw or analyzed data is observed |
