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DNA App. IV

Test 1

QuestionAnswer
Genotyping STR products labeled w/ fluorescent dyes are amplified, seperated and detected.
Peak information Converting the peaks into allele calls.
HID Human Identification.
Genotyping Software ABI users - GeneScan & GeneMapper MegaBACE users - Genetic profiler Gene Marker HID - Does not come w/ machine
Locus-specific allelic ladders Copy of all different alleles present on a locus.
Internal Sizing standards will increase in precision when___? There are more peaks.
Sizing DNA Fragments. Internal sizing standard - GS500-ROX PowerPlex kit - ILS 600 MegaBACE Energy Transfer ROX size standard
Local Southern Method Uses peaks on either sides of the unknown. [(2above)+(2below)]/2
Local Southern Method Issue A big enough range of peaks is needed to make sure there will be 2 peaks on each sides of the unknown.
Global Southern Method Uses all peaks to make a best fit size calibration line.
What can you do to ensure that you are able to compare data over time? Be consistent in the use of standards.
Off-Ladder alleles Alleles the do not fall w/in 0.5bp of an allele using a locus-specific allelic ladder.
How to fix off-ladder alleles? Re-run or re-amplify
What causes off-ladder alleles? Insertion or deletion of 1bp
Microvariant off-ladder alleles Defined as alleles that are not exact multiples of the basic repeat motif or sequence variants.
How are Microvariant off-ladder alleles designated? A whole number for the full repeat + decimal point followed by number of bases in partial repeat
How can you detect "same length but diff. sequence alleles"? By sequencing
What are the 2 types of three-peak patterns? Type 1 - Sum of heights of 2 peaks equal the third one Type 2 - Balanced peak heights
What are the 5 biological artifacts of STR markers? Stutter Non-template nucleotide addition Microvariants Null alleles Mutations
Spike A peak that goes through the 4 channels
Pull-up (bleed-through) Small peaks on channel below following the actual peaks.
Stutter product Peak of 5-15% of the true allele that shows-up one repeat before (rarely after <2%) the true allele caused by slippage during DNA synthesis.
Shorter or longer region have more stutters? Longer
What is the relation between stutters? Each successive stutter is less intense than the previous one.
Non-template Addition Taq polymerase adds an extra nucleotide (A) at the end of the PCR product.
How do you enhance non-template addition? With the extension soak at the end of PCR cycle.
How do you reduce non-template addition? By using a new polymerase.
What will too many RFUs do on the peaks? It will cause them to have a flat end, making them impossible to read.
Null alleles The allele is there but fails to amplify due to a change in the binding site. (allele dropout)
How is imbalance in alleles peak heights occurring? Mutation in the middle of the primer binding site.
How does allele drop-out occur? Mutation at 3' end of primer binding site.
How else, other than the peaks, can you detect mutation? Through the family genealogy
Which loci have higher mutation levels? VWA, FGA and D18S51
Which loci have lowest mutation levels? TH01, TPOX and D16S539
Is it better to promote or prevent non-template addition? Explain. To prevent it because there is less extension time.
What are some approaches to dealing w/ the possibility of allelic dropout due to PCR primer binding site mutation? Amplify the same allele w/ a diff. primer. Test the entire population.
What observations indicate that the peak in an electropherogram is a spike as opposed to an allele? The spike goes through the 4 channels.
What steps are taken to confirm the presence of an off-ladder allele? Re-run or re-amplify product Re-amplify sample Amplify sample w/ single-locus primers
Why are allele sizes different at the same locus between different STR multiplex kits? Because different kits have different ranges.
What is the range of the Mixture Region? 15-70%
Mixture interpretation (1-3) 1. Identify the presence of a mixture 2. Designate allele Peaks (allele VS artifact) 3. Identify the number of potential contributor. (usually 2)
Mixture interpretation (4-6) 4. Estimate he relative ratio of the individuals contributing to the mixture. (Use whole profile/ look a amelogenin) 5. Consider all possible genotypes. 6. Compare reference samples.
Why are mixture interpretation not great in court? Because you are using your opinion instead of facts.
What is the conservative approach? The defendant can't be excluded as a contributor of the mixture.
CPE PE = 2pq + p^2 Combine probability of exclusion
Likelihood Ratio Approach ratio of possibilities under alternative proposition.
Partial profiles Degraded;LCN samples PCR fails to amplify alleles above detection threshold
LCN low copy number DNA low template DNA low level DNA touch DNA Trace DNA
Stochastic fluctuation effect Little amount of DNA causes imbalance between alleles (drop-out or drop-in, high stutter, partial profile.
Possible reason for allele dropout(3) 1. Failure to transfer to cell 2. Target sequence is degraded or not present 3. PCR amplification problems
Enhanced interrogation techniques -Increase PCR cycle number -Reduce volume PCR -Sample desalting prior to CE -Extended CE injections
Enhanced interrogation techniques downside Help but bring up other issues
False Negative Loss of true signal (peak imbalance or allele dropout)
False Positive Gain of false signal (high stutter or allelic drop-in)
Preferential PCR amplification 1. Diff. in GC% between alleles 2. Smaller allelic are amp. preferentially 3. Less efficient priming of DNA synthesis of one allele VS another
Causes of preferential PCR amplification 1. could be due to extension time 2. chemical amounts (e.g. Taq)
LOD Limit of detection: 3x signal-to-noise Lowest [] that can be measured w/ reasonable statistical certainty
LOQ Limit of quantification: Lowest [] that can be determined w/ acceptable precision and accuracy.
How is the possibility of contamination increased w/ LCN? 1 cell can throw off results
2 types of contamination 1. Systematic: it shows up everywhere 2. Sporatic: It shows up in one spot only (it could be anything)
What do you do w/ LCN? increase cycle # increase taq reduce PCR volume longer injections for CE desalt before CE
How to you create a concensus profile? Run PCR in triplicates.
Created by: Kym Boivin Kym Boivin on 2012-01-31



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