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