BIO4320 Molecular Biology & Genetic Engineering Oct.

28, 2003
Name: S.I.D.

ANSWER ALL QUESTIONS

Question 1
The figure on the right shows a diagrammatic representation of the lambda ZAP vector.
During the excision process, XL1-Blue MRF’ and SOLR are employed as the first and
second bacterial hosts, respectively.

(a) What will happen if we wrongly use the SOLR strain

as the first host? (1 mark)
(b) What will happen if we wrongly use the XL1-Blue
MRF’ strain as the second host? (1 mark)
(c) What will happen if the F’ plasmid is lost from the
XL1-Blue MRF’ strain? (1 mark)
(d) What will happen if the ∆M15lacZ region on the F’ is
deleted? (1 mark)
(e) What will happen if the I (initiation site for fl phage
packaging) reciprocally changes position with T
(termination site for fl phage packaging)? ( 1 mark)
(f) Will the lambda ZAP vector better be used to
construct cDNA library or genomic library? Why?
(1 mark)

Question 2 (in PCR reactions) 0%

A student tried to amplify a highly G-C rich DNA DMSO
template using PCR. (Note: ladders on the first and last
lanes of the gels are molecular weight markers. The
single bands on other lanes are the PCR products.) No products

(a) Based on the results shown in the figure on the right,

propose a possible function of DMSO (1 mark)
(b) The student concluded that the FastStart Taq DNA
Polymerase exhibited a higher enzymatic activity than
the regular Taq DNA Polymerase, when G-C rich
templates were used. Explain whether you agree
with the student’s conclusion. (3 marks)

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BIO4320 Molecular Biology & Genetic Engineering Oct. 28, 2003
Name: S.I.D.

Question 3
A patient is infected by a pathogenic bacterium. There are two subtypes of this
bacterium: strain A and strain B. For proper treatments, the doctor needs to check whether
the patient is infected by strain A or strain B.
The genome of strain A is identical to that of strain B except the presence of a 40-bp
region in strain A.

The sequence of the 40-bp region is:

5’-ATCCGGTACATTAGCCGATTGGCGATTTTAACCGGCTTTC-3’
3’-TAGGCCATGTAATCGGCTAACCGCTAAAATTGGCCGAAAG-5’

Suppose a laboratory is provided with the following materials:

(i) The patient’s serum sample that contains only a trace amount (NOT enough for
detection of the pathogenic bacterium by hybridization).
(ii) Four radioactively labeled primers with the following sequences:
Primer A: 5’-ATCCGGTACATTAGCCGATT-3’
Primer B: 5’-GGCGATTTTAACCGGCTTTC-3’
Primer C: 3’-TAGGCCATGTAATCGGCTAA-5’
Primer D: 3’-CCGCTAAAATTGGCCGAAAG-5’
(iii) A heat stable DNA ligase that can seal nicks on double-stranded DNA molecules.
(Remark: NO heat stable DNA polymerase is provided).
(iv) A thermal cycle machine.
(v) Reagents for running a polyacrylamide gel (i.e. able to separate DNA molecules of
small molecular weight).
(vi) X-ray films and the film developing machine.

Design a strategy to diagnose whether the patient is infected by strain A or strain B. Use
diagrams whenever appropriate (7 marks)

Question 4
The same sample from Question 3 was sent to a different laboratory. In this laboratory,
only heat stable DNA polymerase but not heat stable DNA ligase is available. Also, this
laboratory only has reagents for running an agarose gel (i.e. NOT able to separate DNA
molecules of small molecular weight). Other reagents and facilities are the same as that in
Question 1. You can synthesize more primers if needed. Design a strategy to perform the
diagnosis in this laboratory. (3 marks)

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BIO4320 Molecular Biology & Genetic Engineering Oct. 28, 2003
Name: S.I.D.

Question 5
A new 5’-RACE method was invented to specifically target full-length mRNAs. In
eukaryotes, each mRNA contains a poly A tail at the 3’ end as well as a G-cap (m7G-p-p-p) at
the 5’end. A truncated mRNA does not contain a G-cap, but ended with a 5’ phosphate
group (p).

The following enzymes will be used:

(i) CIP (calf intestinal phosphatase): removes 5’ phosphate but not G-cap;
(ii) TAP (pyrophosphatase): removes G-cap to expose the 5’ phosphate group;
(iii) RNA ligase: ligate the 3’-OH group of a RNA molecule to the 5’ phosphate group of a
RNA molecule.

You are provided with a RNA oligo with known sequence.

(a) Draw a flow chart to illustrate the sequence of event leading to the addition of the RNA
oligo to the 5’ end of only full-length but not truncated mRNA molecules). (6 marks)

(b) Propose the subsequent steps for this enhanced 5’-RACE. (Do not forget that you now
have an additional known sequence added to the 5’ end of mRNAs.) (4 marks)

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