Applied Molecular Biology
7.5 ECTS
Ladokcode: 42K02D
The exam is given to: KMAKB14h
Name:
Personal
number:
Date of exam: 13th of January, 2015
Time: 9:00-13:00
Means of assistance:
Accepted aid/tool: Dictionary
NOT accepted aid/tool: Electronic dictionary
Tillåtna hjälpmedel är lexikon. Dock EJ elektroniskt lexikon
Total amount of point on exam: 72 points
Requirements for grading:
Grade A: 65 points ; Grade B: 58 points ; Grade C: 50 points ; Grade D: 43 points ; Grade E:
36points ; Grade FX and F: less than 36 points
Additional information:
Please, answer every question on a new sheet of paper
The results are posted latest three weeks after the exam or at:
Important! Do not forget to write your name on each paper you hand in.
Good Luck!
Examiner:
Telephone:
Elisabeth Feuk-Lagerstedt,
0704-444188
1
Question 1
Which statement is the right one? (2p)
A. The genome of the phage λ is double stranded and circular
B. The genome of the phage λ is single stranded and circular
C. The genome of the phage λ is double stranded and linear
D. The genome of the phage λ is single stranded and linear
E. None of above
Question 2
Which of the following primer would allow copying of the single-stranded DNA sequence
5' ATGCCTAGGTC ? (2p)
A. 5' TACGG
B. 5' CTGGA
C. 5' GGCAT
D. 5' ATGCC
E. 5' GACCT
Question 3
What is a shuttle vector? (2p)
A. A plasmid, which can be integrated into the genome of the host cell
B. A vector that can replicate in cells of more than one type of microorganism
C. A cosmid
D. A cloning vector without genes for selection
E. None of above
Question 4
What is the task of a restriction enzyme? (2p)
A. Fill in nicks in a DNA sequence
B. Cut inside a DNA molecule
C. Ligate DNA fragments
D. Cut a DNA molecule at its ends
E. None of above
Question 5
What is a polylinker? (2p)
A. A double stranded oligonucleotide which can make blunt ends into sticky ends
B. A double stranded oligonucleotide which can make sticky ends into blunt
C. A double stranded oligonucleotide which has a restriction site for one restriction
enzyme
D. A double stranded oligonucleotide which has a restriction site for many restriction
enzymes
E. None of above
Background information for question 6-11:
Agaricus biosporus is an edible mushroom able to produce the enzyme tyrosinase often
referred to as polyohenoloxydase (PPO). This enzyme is able to oxidize various phenolic
compounds and is therefore an important for a number of biotechnological applications.
The fungal tyrosinase-coding PPO2 gene was isolated and amplified by RT-PCR using total
RNA extract from the mushroom fruit bodies. (The DNA was sequenced before the PCR
step). The PPO2 gene was then cloned into YEp24 and transformed into an appropriate host
cell. The recombinant molecule was later isolated and finally transformed into S. cereviciae.
Recognition sites for some restriction enzymes
AhdI: GACNNN^NNGTC
SmaI: CCC^GGG
ApaI: GGGCC^C
SnaBI: TAC^GTA
AseI: AT^TAAT
SphI: GCATG ^ C
BamH1: G ^ GATCC
XbaI: T ^ CTAGA
NcoI: C^CATGG
Please, answer every question on a new sheet of paper!
Question 6
To be able to clone the gene into the vector you first have to produce ds cDNA copies of
isolated RNA.
Describe the method you are using and explain each step. (8p)
Question 7
The ds cDNA sequence encoding POP2 was amplified by PCR and the following pair of
specific primes with restriction sites was used:
Forward: 5’- AAAAGGATCCTCCGACACTGCCTTGATC- 3’
Reverse: 5’-AAAATCTAGATTCCACTGGGCGATGATG- 3’
Explain the amplification process and motivate each step. (9p)
Question 8
It is now time to insert the amplified gene product into YEp24.
a) Which restriction enzyme/enzymes will you use? Give motive for your answer! (6p)
b) Which other enzyme/enzymes do you use to create the recombinant molecule? (2p)
Question 9
When you have created the recombinant DNA molecule you want to transfer it into your host
cell to get a clone carrying many copies of your recombinant molecule.
a) What type of host cell do you use in the first transformation process? Give motive for
your answer! (4p)
b) Which specific qualities does the host cell have? (4p)
c) Explain how you carry out the transformation and mention the purpose of each step.
(6p)
Question 10
After the transformation you do a selection process and you find a clone carrying the POP2
gene. To be able to transform the recombinant vector into a yeast strain instead you first do a
plasmid preparation. Describe this process and the motive of each step. (10p)
Question 11
Before you can transform S. cerevisiae you must do an overnight culture of the yeast strain
and grow it until early log phase. Explain an easy method to follow the growth. (5p)
Question 12
After the transformation process of an appropriate strain of S. cerevisiae you want to find
recombinants carrying the Pop2 gene. Explain how you are doing this and the purpose of each
step. (8p)