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FRNSC 421W - EXAM 1-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 421W - EXAM 1-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 421W - EXAM 1-Questions with Correct Answers/ Verified/ 100% PassRFLP - ✔️✔️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
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
DQα - ✔️✔️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 H₂O₂ 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:
◦ [7(7+1)]/2 = 28 theoretical genotypes for DQα
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
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
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
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
, 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
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 temp
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 quality
non-qPCR (ex, agarose gel):
- Fast, crude, relatively inaccurate
- Cannot use low-quality or degraded samples
- Not human-specific
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)
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