BIOC301 – Xmas Review Problem Set
Q1. Which of the following primers would allow for extension of the single-
stranded DNA molecule 5’-GTACTAACCGA-3’ ?
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’
Ina OVR-h 5’-ATGGTACTAACCGAGATGCCGCAAATAGATTAA-3’
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.
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
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
94 / 7min
94 / .5min
58 / .5min
72 / 1min
Repeat previous 3 steps
! 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
!" ! !! !"""!" !"!
ANS: 100 !∙ ∙ ∙ =
!" !" !"""!" !! !
! ∙174.2! = 0.05741!!"#/!
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)
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.