FRNSC 421W - EXAM 1 PRACTICE QUESTIONS AND ANSWERS (100% PASS)
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Course
FRNSC421W /FRNSC 420
Institution
FRNSC421W /FRNSC 420
FRNSC 421W - EXAM 1 PRACTICE QUESTIONS AND
ANSWERS (100% PASS)
RFLP - Answer️️ -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...
DQα - Answer✔️✔️-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? - Answer✔️✔️-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 - Answer✔️✔️-hybridization and end point PCR assay; relatively
accurate, expensive
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 - Answer✔️✔️-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 - Answer✔️✔️-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
3
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