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DNA App. IV
Test 1
| Question | Answer |
|---|---|
| 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:
kboivin
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