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BIO4320 Molecular Biology & Genetic Engineering Name: S.I.D.

Oct. 28, 2003

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)

(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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(in PCR reactions)

0% DMSO

No products

Question 2 A student tried to amplify a highly G-C rich DNA 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.)

BIO4320 Molecular Biology & Genetic Engineering Name: S.I.D.

Oct. 28, 2003

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 Name: S.I.D.

Oct. 28, 2003

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) (ii) (iii)

CIP (calf intestinal phosphatase): removes 5’ phosphate but not G-cap; TAP (pyrophosphatase): removes G-cap to expose the 5’ phosphate group; 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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