Miscellaneous Flashcards
What does dNTP stand for?
- deoxynucleoside tri-phosphate
- 5-carbon sugar bound to a nitrogenous base with three phosphate groups covalently bound to the sugar
What is the size of the mtDNA genome?
~16,569bp
What is the purpose of the 56C incubation in chelex extractions?
-allows for chelex to bind polyvalent cations
What is the purpose of the boiling step in chelex extractions?
-To lyse cells, denature DNA and destroy proteins
Does anything affect ion binding ability of Chelex?
-pH; it works better when pH is between 10-11
What types of controls are used in processing? What stages are they initiated?
Extraction reagent blank – at start of extraction (SOP?) sample prep (sanding/grinding); Chelex at beginning of extraction
Amplification RB, Neg 1, Neg 2 – amplification
Is DNA always found in the aqueous layer of an organic extraction?
-once it has been spun to have layer separation, yes
What is the role of EDTA?
- it chelates Mg2+ cofactors to inactivate DNAases
- helps break down the hydroxyapatite matrix to make more DNA available
- preservative
What is the molecular classification of a nuclease? Why is this important with regards to DNA extractions?
-nucleases are protein enzymes that degrade DNA to reuse components. In an extraction, they destroy DNA.
What is meant by a serine protease?
-it breaks down the serine amino acid residue at active site of enzymes
What volume(s) of Demineralization buffer is(are) used for teeth in both organic and non-organic extractions?
-4mL organic, 4mL or 7.5mL non-organic
What are the spin parameters for the first Ultra4 spin for bone and tooth organic extractions?
-2000G for 40-50min(bone); 30-40min (tooth)
If a sample will not concentrate to an appropriate volume in an organic extraction, what steps(s) should be performed?
-If the desired volume has not been reached then the Ultra4’s should be continued to be spun. If this does not work then a new ultra4 may be used
What is the 30kDa cutoff for ultra4-30ks referred to as?
-nominal molecular weight limit
What charge is silica?
-negative
What type of bond is formed between DNA and the silica membrane of the QIAquick column?
-salt bridges
What does chaotropic mean?
-disrupting the structure of hydrogen bonds in molecules
What is the rnage of recovery in the Demin II extraction?
50-200uL
Besides ethanol, can anything else be used to clean spills from non-organic extractions?
-a suitable laboratory detergent and water
What are the possible results for samples in an extraction yield gel?
-band and/or smeaer or no band and/or smear
What is meant by deamination?
-the amino group is lost through hydrolysis
What is the name of the codes used to signify point heteroplasmy?
-IUPAC nucleotide codes
When should an “N” be reported for insertions in length heteroplasmy?
-when the predominant species cannot be identified
How many capillaries are on a 3500xL?
-24
When are spatial calibrations performed?
-weekly or when the capillary is changed or removed for any reason
What needs to be included, if anything, if an RB or NEG needs to be re-Exo’d? Re-sequenced?
-all samples and associated controls must be re-Exo’d and/or re-sequenced
What needs to be included if a sample needs to be re-injected?
-when reinjecting for an increased time the negative, RB, and substrate control(if applicable) must be included; otherwise if the time remains the same, just whatever sample is being re-injected
What inhibitors are found in soil?
-humic and fluvic acid
Besides “consistent with” what are the remaining reporting criteria for 12S?
-presumed to be and inconclusive
Describe the parameters for CPD searches and the purpose for each parameter selected.
-display only up to ‘n’ differences = 0
-use partially overlapping profiles
-ignore general insertions
-restrict regions
-CPD databases
The purpose is the ensure highly polymorphic areas that can change between individuals of the same lineage are not included. Including these areas would likely exclude a reference as a family member due to their highly variable nature since we commonly do not have a 1 to 1 match
What parameters are selected for staff CPD search?
- display only up to ‘n’ differences = 0
- use partially overlapping profiles
- ignore 16193.1C
- staff sequence database
What is the purpose of Clopper Pearson? Why are likelihood ratios calculated this way?
- confidence interval for our calculations
- we calculate LRs this way in order to give a more conservative value for infrequent profiles
What CPD populations are used for calculating likelihood ratios on a BTB?
- African American
- Caucasian
- Hispanic
If there is no documentation of an individual’s race, what likelihood ratio gets reported in the final statement?
-The most conservative among population groups tested
What wording is used when communicating the likelihood ratio in the BTB report final statement?
-The genetic data(mtDNA) are approximately XXX times more likely to be observed under the scenario that CIL sample ## originated from a maternal relative of (reference name) as opposed to CIL Sample ## originating from an unrelated individual from the xyz population.
What is the “transposed conditional” and give an example of it in a case where the estimated frequency of a DNA profile is 1 in 273?
- when the statistaic is presented in a way that speaks about circumstances not about the evidence.
- Instead of “it is approximately 273 times more likely to be observe the genetic data under the scenario that the sample originated….”, it would be flipped to say “it is approximately 273 times more likely that this is the service member as opposed to a random member of the population.”
What accreditations does AFDIL hold? Does AFDIL get re-accredited and if so, how often?
- FBI QAS (Quality assurance standards for forensic DNA testing laboratories”
- ANAB AR 3125 “ISO/IEC 17025:2017 - forensic science testing and calibration laboratories accreditation requirements”
- re-accreditation is every 4 years with surveillance on the off years; FBI check every 3 years
What types of beads are Chelex beads?
-silica beads
Was the rCRS generated for the CR or the entire mtDNA genome?
-entire mtDNA genome
Give an example of a transition and transversion.
- Transition: T-C
- Transversion: T-G
Why can we not use IUPAC heteroplasmy codes for negatives and RBs?
-we don’t/can’t confirm them so to call it a heteroplasmy would not be the most conservative call
When interpreting length heteroplasmy in the HV2 C-stretch, is an “N” insertion always warranted? Explain why or why not.
-No, calling and N insertion is warranted only when the predominant species cannot be identified.
How do you report the C-stretch in the HVIII region?
-Report the first 6 C’s present and cut additional data
Briefly describe what you do if you receive a comparison or BTB report request.
- comparison: go into LISA and use batch pairwise function. Select comp req to set default parameters. Use match criteria to determine results of comparison - only comparing overlapping regions.
- BTBs, gather all relevant files, rerun comparisons, run g:cats, summary matrix report, and then use LSAM to get likelihood ratio. Then fill out remplate with information gathered
What is the point of the blind hit g:cat? What do you search your sample against and why?
-A blind hit g:cat searches against the FRS database using the conflict of interest and unknown check boxes to filter results. The sample is searched this way in order to compare a sample to only the reference samples that may be associated with those remains.
Would the following sequence come up as a results/hit in sample’s staff and CPD g:cat searches?
Staff: 16024-16569 263G, 309.1C, 315.1C
Sample: 16024-16391, 35-369 263G, 315.1C
Staff: no b/c we look for identical sequence and don’t restrict regions/ignore general insertions
CPD: yes b/c we restrict regions/ignore general insertions so 263G matches
Would the following sequence come up as a results/hit in sample’s staff and CPD g:cat searches?
Staff: 16024-16569, 1-576 16093C, 263G, 315.1C
Sample: 16024-16391, 35-369 263G, 315.1C
No for both b/c the sample does not have the 16093C and the parameters for both types of searches include this position
Using mtDNA match criteria, determine consistency between the sample and FRS and give a reason for your answer:
FRS 16024-16569, 1-576 16093C, 73G, 263G, 315.1C
Sample: 16024-16391, 35-369 73G, 263G, 315.1C
-consistent b/c there is no differences in overlapping profile other than 16093C, which we exclude due to its highly heteroplasmic nature
Using mtDNA match criteria, determine consistency between the sample and FRS and give a reason for your answer:
FRS 16024-16569, 1-576 16519C, 73G, 263G, 315.1C
-cannot be excluded/inconclusive b/c 16519C is not in overlapping regions, 73 is a difference, 309 and 315 are excluded due to being part of the HVII C stretch, which is highly heteroplasmic in nature. One difference is inconclusive
You are doing second analysis on a hair case, the results of which are listed below. Is the 01A1.1 data usable? Why or why not RB1.1: no data generated/clean RB2.1: 150N, 195N 01A1.1: 195C 02A1.1: 150T
The data from the 01A1.1 amplification may not be used b/c it matches RB2.1
Once a case is submitted for review, all changes to examination records must be tracked. Which method is used for the follow?
g:cats, mtDNA case summary sheets, pen and ink changes AND electronic changes, N/A
g:cats - N/A mtDNA case summary sheets - pen and ink changes only H Case data summary (.pdf) - N/A BTB reports - N/A SOP: document control
What are the parameters for printing electropherograms in Sequence Analysis
CR: 6 panels/ 1500 pts per panel
HV: 5/1200
MPSs/PSs: 4/1000
What does NWR stand for? Which SOP is followed for NWRs? Who can request NWRs?
- nonconforming work review
- The Management Improvement System
- Any staff member may notify a supervisor or Key Management. They will asses the situation and take action to correct the nonconformance
Extraction yield gel
- want to see high MW band for DNA with faint smear for bacteria (smear and band will appear super bright and overblown if lots of bacteria present)
- 123bp ladder (NOT mass ladder) - only shows size, not quantity of DNA present
