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)
No λ or fl helper phage infection.

(b) What will happen if we wrongly use the XL1-Blue

MRF’ strain as the second host? (1 mark)
fl helper phage continues to propagate and being secreted
into the growth medium.

(c) What will happen if the F’ plasmid is lost from the

XL1-Blue MRF’ strain? (1 mark)
No F pili and hence no fl helper phage infection.

(d) What will happen if the ∆M15lacZ region on the F’ is

deleted? (1 mark)
Cannot perform blue-white screening even in the
presence of X-gal and IPTG.

(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)
The fl phage coat will pack another strand of the DNA
flanked by I and T. After all subsequent processes,
double-stranded plasmids will be formed but the clone
will be in opposite orientation.

(f) Will the lambda ZAP vector better be used to

construct cDNA library or genomic library? Why?
(1 mark)
cDNA library is better because the plasmid portion
occupied some of the packaging capacity.

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

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)
DMSO may help to reduce the annealing at G-C rich
regions and hence allows better PCR.

(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)

The conclusion may not be correct (0.5), since PCR

products cannot be formed without DMSO, despite of the
type of Taq enzyme used (1). DMSO may have some
inhibitory effects to the enzymes (1). The FastStart Taq
enzyme may be more resistant to these inhibitory effects
by DMSO (1).

If the answer is “agreed”, check to see if valid reasons

were given.

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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)

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

(1) Prepare DNA sample (0.5);

(2) Mix DNA sample (0.5), all four primers (0.5), and heat stable DNA ligase (0.5);
(3) Heat to denature double-stranded DNA template (0.5) and then anneal the primers to the
DNA templates (0.5);
(4) The DNA ligase will seal the nick between two primers that hybridize side-by-side to the
template DNA (1);
(5) Primer A + Primer B and Primer C + Primer D will become Primer A–Primer B and
Primer C–Primer D fragments, respectively (1);
(6) Primer A–Primer B and Primer C–Primer D fragments will become templates for the
ligation reaction in the next cycle (1);
(7) Repeat the cycle of heating, annealing and ligating (1);
(8) Run on polyacrylamide gel, Southern blot, detection by X-ray autoradiography (1);
(9) If the sample gives a 40-bp band, it contains strain A (1).

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)

(1) Choose one primer from A, B, C, or D (1);

(2) Synthesize another primer away from the 40-bp region (1) and this new primer should
orient in an opposite direction to the primer chosen above (0.5);
(3) Run on agarose gel and perform Southern blot to detect DNA band of expected size (1);
(4) If the sample gives positive results, it contains strain A (0.5).

Remarks: if use two primers flanking the 40-bp region, give 1 mark only (since the
differences resulting from strain A and strain B cannot be seen using agarose gel.

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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)
(1) Add CIP to remove 5’ phosphate groups from truncated mRNA (1);
(2) Add TAP to remove G-cap (1) and expose 5’ phosphate groups from full-length mRNAs
(1);
(3) Add RNA oligo and RNA ligase (1);
(4) RNA oligo will only ligate to full-length mRNAs but not truncated mRNAs (1) because
truncated mRNAs do not 5’ phosphate groups (1);

(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)

(1) Reverse transcription using GSP1 and reverse transcriptase (1);

(2) RNaseH to remove mRNA (0.5);
(3) Making second strand using a primer designed based on the sequence of the RNA oligo
(0.5);
(4) First round PCR using GSP2 and a primer designed based on the sequence of the RNA
oligo (1);
(5) If necessary, second round PCR using nested GSP and a primer designed based on the
sequence of the RNA oligo (1).

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