BIOC301 – Xmas Review Problem Set ANSWERS(2).pdf

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Department
Biochemistry
Course
BIOC 301
Professor
Jason Read
Semester
Spring

Description
BIOC301 – Xmas Review Problem Set Q1. Which of the following primers would allow for extension of the single- stranded DNA molecule 5’-GTACTAACCGA-3’ ? a) 5’-CATGAT b) 3’-CATGAT c) 5’-AGCCAA d) 5’-TCGGTT e) None ANS: In order to extend DNA a free 3’-OH must be present after a DNA duplex. The primer must then be complimentary to the given oligonucleotide’s 3’ end. Nucleic acid synthesis always occurs 5’ ! 3’, as the 3’-OH of ribose (nucleophile) attacks the phosphorus atom of the incoming dNTP or NTP (DNA or RNA, respectively). Q2. Which of the following primers would allow for extension of the single- stranded DNA molecule 5’ –GCGGCCGC- 3’ ? a) 5’ – GCGG b) 3’- GCGG c) 5’ – GGCG d) 3’ – GGCG e) More than one choice ANS: As above, any DNA duplex resulting from the annealing of a primer with the given oligo such that a 3’-OH sits against a 5’ single-stranded overhang can and will be extended by DNA Polymerases. Q3. The overshare-hydrolase(OVR-h) enzyme is inactivated in people who LUV fb & twitter. You postulate that this inactive (ina) form is different from the normal, wild-type (wt) form at the gene level and thus design an experiment to check this. Using the gene information below write out the primers you will use in your PCR reactions, 8mers should be sufficient here. wt OVR-h 5’-ATGGTACTAACCGAGCGGCCGCAAATAGATTAA-3’ 3’-TACCATGATTGGCTCGCCGGCGTTTATCTAATT-5’ Ina OVR-h 5’-ATGGTACTAACCGAGATGCCGCAAATAGATTAA-3’ 3’-TACCATGATTGGCTCTACGGCGTTTATCTAATT-5’ Primer 1 (5’!3’): ___ATGGTACT____ Primer 2 (5’!3’): ___TTAATCTA____ ! 1! Knowing that the restriction endonuclease NotI cleaves after the first GC of the palindromic sequence 5’-GCGGCCGC, describe an experiment which would allow you to differentiate between inactive and active forms STARTING FROM PURIFIED DNA SAMPLES. Purified!DNA!!!PCR!amplify!fragments!of!interest!from!all!samples!!!Clean!PCR! sample!(OK,!if!this!was!left!out!for!this!level)!!!Restriction!Endonuclease! Digestion!with!NotI!!!Analyze!samples!by!electrophoresis.! ! The!mutated!version!lacks!the!NotI!site!and!should!consequently!show!up!as!1! band!of!distinct!bp!length!while!the!wildKtype!gene!will!be!cleaved!once!by!NotI,! resulting!in!2!bands!of!distinct!length.! ANS: The idea here is to recognize that the wild-type and mutated version differ in cleavage sensitivity by NotI. In order to access this, one must first obtain a pure sample of a large amount of each nucleic acid. How do we specifically amplify a lot of nucleic acid out of a complex (3 Mbp human genome) sample? PCR of course! This experimental set-up is often used in biomedical sciences to quickly genotype an individual. Small sequence differences genome-wide can be identified between individuals using restriction fragment length polymorphisms (RFLP). Q4. Your instructor has assigned you to clean up after a lousy TA who did not save PCR programs. You are to program the thermocycler so the BIOC420 class can proceed with their experiments this afternoon. In his notebook, you find some chicken-scratch and decipher the following: 72 / 1min 58 / .5min 94 / .5min 94 / 7min Repeat previous 3 steps 40x You suspect this information is out of order and re-input correctly into the thermocycler: 94 / 7min 94 / .5min 58 / .5min 72 / 1min Repeat previous 3 steps 40x ! 2! ANS: This is a 3-step PCR program. Initially a very high temperature is held for a long period of time to fully melt the DNA samples in solution (ie. dsDNA ! ssDNA). After the initial denaturation, 3 steps are repeated in each cycle: denature-anneal-extend. The annealing stage allows the primers to anneal to their complimentary sequences while the extending stage is simply the temperature at which Taq Polymerase (& other polymerases used in PCR) achieves maximal activity. It is important to note that although described in an exclusive fashion, these steps are not exclusive: As soon as the 3’ end of a primer has “sat down” in the annealing stage Taq Polymerase can extend it. It simply does so more slowly at 58º vs. 72º. Q5. A 1:10 solution of 100.0mg/mL PMSF (MW 174.2) is of what molarity? Show unit analysis. !" ! !! !"""!" !"! ANS: 100 !∙ ∙ ∙ = !" !" !"""!" !! ! 10! 1!"# ! ∙174.2! = 0.05741!!"#/! 0.05741 M Q6. You are volunteering as an undergraduate in Scott Covey’s lab. Because you successfully gone through the BIOC301 lab, he is confident you can make all of the solutions for his experiments. How would you make the following solutions? Note: you only have access to a weight-based scale; you CANNOT weigh out in mole units! a) 1L of 100mM Tris-HCL (121.14 g/mol) b) 200mL of .5M MgAc (110.39 g/mol) 2 c) 100μL of 1x Protease Inhibitor Cocktail (Stock from Roche Scientific is 25x) d) 416μl .25mM NADH (30mM stock solution) ANS: a) Disolve 12.1g of Tris-HCl in dH O 2nd make up the volume to 1L. b) 11.0g MgAc in2dH O a2d make up the volume to 200mL. c) Add 4ul of 25x stock to 96ul of dH O. d) 3.47ul of 30mM stock to 412.53ul dH O. 2
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