FRNSC 421W - EXAM 1 100 QUESTIONS
AND ANSWERS 2024
1 RFLP: restriction fragment length polymorphism
- DNA digested by restriction enzymes at repeat sequences, yields different
fragment lengths for different people
- Gel electrophoresis to separate fragments by size, MW ladder for reference
- Southern blotting to physically transfer DNA to membrane
- Radioactively labeled probe hybridizes with immobilized DNA on membrane-
Exposure to X-ray film yields carbon copy of gel, reveals unique blotting pattern
specific to genotype
- Single locus probes use 1 probe to hybridize with multiple loci; only need 4-6 loci
to support a match
- Labor intensive but highly discriminatory; requires high MW and high-quality
DNA
2. D1S80: highly variable VNTR locus, separates DNA fragments by allelic
number (smaller fragments/allele # migrate further)
- Resolves alleles of loci and runs on a silver-stained gel; allelic ladder represents
known # of repeats
- DNA extraction and PCR amplification with silver staining instead of
radioactive/fluorescent probes
- can use on lower quality and amounts of DNA than RFLP
3. DQ±: gene on chromosome 6 that codes for cell surface receptor involved in
immune response, SNP-based (PCR) reverse dot blot assay
- Immobilized probe fixed to nylon membrane by tails; PCR product hybridizes
to probe
- PCR primers have biotin group that binds to Streptavidin-horseradish
peroxidase complex
- upon binding to biotin, SA-HRP catalyzes oxidation of 3,3',5,5'-
tetramethylbenzidine (TMB, chromogen) in the presence of H‚O‚ to generate blue
precipitate (chromophores) on membrane = colorimetric quant.
- anywhere that probe binds target, SA-HRP complex binds and yields blue
dot on nylon strip
, .
- Tests for 7 alleles at 1 locus, not very informative on its own- given that
[n(n+1)]/2:
æ [7(7+1)]/2 = 28 theoretical genotypes for DQ±
4. why is quantification important?: 1) It's a required standard associated with
accreditation by SWGDAM, FBI, ANAB, etc.
2) Protects integrity of DNA extract (a form of evidence)
3) Enhances the possibility that extracted DNA will be available for defense to
perform independent testing
4) Better allows for appropriate amount of DNA so downstream methods are more
successful; very narrow window of [DNA] required for sufficient results
5 Quantiblot: hybridization and end point PCR assay; relatively accurate,
expensive
- all levels of DNA quality, human specific
1) small sample of extracted DNA is deposited and fixed on nylon membrane
(RLFP path)
2) biotinylated human probe is hybridized to DNA fixed on membrane
3) through colorimetric/chemiluminescent-based process, intensity of developed
image = quantity of DNA in extract
- more DNA = more probe binds = brighter report
- amplifies a target and assumes other samples are equal
6. Quantifiler: real-time qPCR assay
- Targets untranslated, intronic hTERT (human telomerase reverse transcriptase)
gene sequence for autosomal DNA and Y-chromosomal DNA for sex
determination - After each round of PCR, a tungsten-halogen lamp excites free
reporter dye and fluorescence emission captured with CCD camera
- Digitally graphs fluorescent signal over cycle #, allows for estimation of DNA
quantity
- All levels of DNA quality, human-specific; 2x as sensitive as Quantiblot
- considered a TaqMan assay
7. Quantifiler DNA quantification procedure: 1) make quantification standards;
five 10-fold dilutions of 50 ng stock
2) make master mix (from # of rxns x 8µL primer mix and 10µL PCR reaction mix),
add 18 µL to each applicable well
3) add 2 µL of each standard to respective well
, .
4) add 2 µL of each sample and RB (RB should be added last); add no additional
volume to 18 µL mix of NTC
5) seal reaction plate and ensure no bubbles
6) place tray in AB 7500 and run reaction through 7500 system software
7) evaluate results of standard curve and reaction; export or print quant data
8. TaqMan: qPCR assay
1) minor groove binder (MGB, or the probe) anneals DNA downstream of PCR
and stabilizes binding of reporter probe
2) MGB binds non-fluorescent quencher (NFQ) on 3' end of reporter probe
3) NFQ quenches reporter fluorescent dye on 5' end of probe
4) dye absorbs energy & emits fluorescence; not visible, quencher absorbs
5) Taq polymerase acts as exonuclease, cleaving probe
6) dye is now unquenched and will fluoresce
- intensity of fluorescence = amount of DNA
9 minor groove binder (MGB): anchors to minor groove of DNA; binds quencher
of reporter probe to allow for fluorescence - interacts through vdW forces and H-
bonds
- planar configuration allows for intercalation, fits into smaller sequences
- DNA portion of reporter probe much smaller than most PCR primers (~12 bases)
- acts as a 27mer, increases melting temp10. qPCR advantages: qPCR:
- More accurate, but can be time-consuming and expensive
- Human-specific, Quantifiler is Y-chromosome specific
- Can use all levels of DNA qualitynon-qPCR (ex, agarose gel):
- Fast, crude, relatively inaccurate
- Cannot use low-quality or degraded samples
- Not human-specific
11. qPCR amplification: amplification of loci with unlabeled primers and
fluorescently labeled probes
- Product is unquenched dye through digestion of probe that binds target region
between PCR primer pair; digestion of probe releases dye from quencher,
allowing for fluorescence (TaqMan assay)
- Tungsten-halogen lamp excites free reporter dye and emissions are captured by
CCD camera
- Digitally graphs fluorescent signal over cycle # while the reaction is taking place
(i.e., in real time)
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