BN 212: MOLECULAR BIOTECHNOLOGY FINAL EXAMINATION: 2011/2012

BN 212: MOLECULAR BIOTECHNOLOGY

FINAL EXAMINATION: 2011/2012

      DATE: 31^ST January 2012                                                                         TIME: 14:00-16:00h

INSTRUCTIONS: ANSWER ALL QUESTIONS FROM SECTION A AND ANY TWO QUESTIONS FROM SECTION B (TOTAL 60 MARKS)

SECTION A (30 MARKS)

1)   In the DNA sequence below, the positions of two oligonucleotides are marked:

a)    What is the melting temperature (T_m) for each of these oligonucleotides?

The formulas used is Tm = 4(G+C) + 2(A+T)

For the left hand primer, Tm = 4(8+3) + 2(1+7)

                                                  =44+ 16

                                                  = 60⁰C

For the right hand primer, Tm = 4(4+5) + 2(5+7)

                                                    =36 +24

                                                    =60⁰C

b)   What length would the polymerase chain reaction product primed by these two oligonucleotides be?                                                                           (6 marks)

2)       Briefly explain how bacterial cells can be made competent for a transformation experiment.

Ø Electroporation;

An alternative to chemical treatment of bacterial cell to make competent cells is called electroporation. In this process the bacteria are harvested and washed as before but in cold distilled water or a buffer with a very low ionic strength. A small sample of a dense suspension of these bacteria is then

mixed with the DNA in a special cuvette and a short pulse of a very high voltage current is passed through the bacterial suspension. Again, it is not entirely clear why this technique works but it is thought that the high voltage pulse makes the bacterial membrane more permeable and possibly moves the DNA into the bacterium by a process akin to electrophoresis (Section 3.11). Electroporation is a much more efficient process than chemical transformation and it can be used with bacteria other than E. coli. Electroporation can also be used to transform mammalian and plant cells

Ø Chemical treatment;

To prepare competent E. coli a culture is grown and then harvested when it is in log phase, at which stage the bacteria are dividing rapidly. The cells are harvested by centrifugation and washed several times in a chilled buffer containing divalent cations, typically CaCl2. The bacteria are finally suspended in a small volume of the buffer so that they are present at a high density. To introduce DNA (for instance, a recombinant plasmid molecule) into these cells, a small sample of this suspension is mixed and incubated on ice with the ligation mixture; it is then heat-shocked at 42°C for about

1 min. This technique has been widely used for many years, although the precise mechanism by which it causes E. coli cells to take up DNA is only now becoming clear.

Ø Gene gun;

One of the most effective techniques is to shoot microprojectiles coated with DNA into plant and animal cells. The gene gun, first developed at Cornell University, operates somewhat like a shotgun. A blast of compressed gas shoots a spray of DNAcoated metallic microprojectiles into the cells. The device has been used to transform corn and produce fertile corn plants bearing foreign genes. Other guns use either electrical discharges or high-pressure gas to propel the DNA-coated projectiles. These guns are sometimes called biolistic devices, a name derived from biological and ballistic.

3)       Why are the following steps important in a cloning experiment?

a)            Inactivation of restriction enzymes before adding the DNA ligase.

Ø In order to avoid the cleaving the ligated sites that are the ligation between the plasmid and the new strand as the R.E are responsible.

b)           Inactivation of alkaline phosphatase before mixing the vector and the fragment insert.       

Ø To avoid the re-circulization of the plasmid as the alkaline phosphate group can join with OH group (6 marks)

4)       Account for the use of the following chemicals in polyacrylamide gel electrophoresis:

a)    β-Mercaptoethanol and Dithiothreitol

Ø The purity and molecular size of a protein can be assessed by analyzing proteins in the presence of 0.1% SDS on one-dimensional gels (UNIT 8.4) or two dimensional gels (UNIT 8.5). By adding a reducing agent (e.g., 2-mercaptoethanol or dithiothreitol) to the sample, it is possible to determine the number and size of subunits in a pure protein. Non denaturing gels can be used to examine and isolate the “native” protein.

b)   Sodium dodecyl sulphate (SDS)

Ø Is a detergent that coats and solubilize the protein from quaternary structure to primary structure that is movement is according to molecular size only (not mol. Charge)

c)    TEMED

Ø Is an enzyme that catalyses the formation of free radical from ammonium persurphate.

d)   Ammonium persulphate  

Ø Is a catalyst that initiates the polymerization of acrylamide and bis acrylamide during PAGE(6 marks)

5)   a) What is DNA profiling?

Ø Is a method of determining the specific genome of a human individual which discriminate from other humans.

      b) What is the advantage of using short repeat length (STRs) over long repeat length simple tandem repeats for forensic analysis?

Ø These STR loci are highly polymorphic with a wide variability in the number of repeats between individuals compared to long repeats.

     c) Why should STR loci selected for forensic profiling be distributed throughout the genome?

Ø In choosing STR loci for forensic profiling it is also important that they are distributed throughout the genome (Table 13.1) to ensure that each is inherited independently.

SECTION B (30 MARKS)

6)   Describe how successful ligation assay may be detected by insertional inactivation using:

a)    Plasmids like pBR322 with more than one antibiotic resistance gene.

Ø A plasmid like pBR322 has both ampicillin resistance gene and tetracycline resistance gene that allow the growth of bacteria when grown on them respectively. In this technique only one of these is used for instance if ampicillin resistance gene is used, the new insert is inserted on the ampicillin gene that interfere the resistance of the bacteria when grown on the ampicillin hence cause a bacteria not to grow, therefore those which fail to grow means insertional inactivation is successful. And when tetracycline is used the new insert is inserted at the tetracycline resistance gene that destroys its resistance that cause the death of the bacteria when grown on the tetracycline therefore those dies are the that are successful transformed.

b)   Plasmid like pUC18 and its derivatives which make use of a gene called lacZ.

Ø Within the lac Z gene of a pUC18 plasmid there is multiple cloning site (MCS) which codes for an enzyme β-galactosidase. β-galactosidase converts a substrate known as X-gal/IPTG to bright blue product that is when cells are grown on an agar plate they produce a blue colonies       therefore when the new insertion is successful it disrupts the lac Z that it becomes unable to code for the X-gal/IPTG hence it do not produce the blue colonies but the normal color that is white is produced. Therefore in order to know that the insertional inactivation is successful white colonies are produced when grown on an agar plate.                                                                                           (15 marks)

7)   There are many ingenious ways of identifying the clones that contain the cDNA of interest from a cDNA library. Explain how this can be done if:

o   We will describe two ways of selecting cDNA clones from a library. One method simply detects the presence of the foreign DNA attached to the plasmid vector, and the second detects the protein encoded for by the foreign DNA. We call the process of selecting specific clones “screening the library”.

Ø Preparation of the cDNA Library for Screening.

a)    The clone is to be selected by virtue of its DNA sequence.

Ø Bacterial colonies are plated onto agar plates, and the colonies are then replica-plated onto a nylon membrane, which is then treated with detergent to burst (or lyse) the bound cells. If the clone is to be selected by virtue of its DNA sequence (Fig. 7.7), the nylon membrane is processed with sodium hydroxide. This is necessary to break all hydrogen bonds between the DNA strands bound to the nylon membrane and ensures that the DNA is single-stranded. The processed nylon membrane is an exact replica of the DNA contained within each bacterial colony on the agar plate.

b)   The clone is to be selected by detecting the protein encoded by the foreign DNA.                                            (15 marks)

Ø If the clone is to be selected from the library by detecting the protein encoded by the foreign DNA (Fig. 7.8), then colonies are again replica-plated on to a nylon membrane. This time, however, the nylon membrane is processed to produce an exact copy of the proteins synthesized by each bacterial colony.

8)   a) What must be done in order for a cloned gene to be expressed in a host bacterial cell?

Ø An expression vector must be used.

Ø The recombinant gene must have a promoter that is recognize by the host RNA polymerase.

Ø The prokaryotic leader must be provided to synthesize eukaryotic protein in a bacterium.

Ø Introns in the eukaryotic gene must be removed because the prokaryotic host will not excise them after transcription of mRNA.

      b) Outline the procedure for Somatostatin cloning and production.                     (15 marks)

Ø Somatostatin, the 14-residue hypothalamic polypeptide hormone that helps regulate human growth, provides an example of useful cloning and protein production. The gene for somatostatin was initially synthesized by chemical methods. Besides the 42 bases coding for somatostatin, the polynucleotide contained a codon for methionine at the 5’ end (the N-terminal end of the peptide) and two stop codons at the opposite end. To aid insertion into the plasmid vector, the 5’ ends of the synthetic gene were extended to form single-stranded sticky ends complementary to those formed by the EcoRI and BamHI restriction enzymes. A modified pBR322 plasmid was cut with both EcoRI and BamHI to remove a part of the plasmid DNA. The synthetic gene was then spliced into the vector by taking advantage of its cohesive ends. Finally, a fragment containing the initial part of the lac operon (including the promoter, operator, ribosome binding site, and much of the β-galactosidase gene) was inserted next to the somatostatin gene. The plasmid now contained the somatostatin gene fused in the proper orientation to the remaining portion of the β-galactosidase gene.

After introduction of this chimeric plasmid into E. coli, the somatostatin gene was transcribed with the β-galactosidase gene fragment to generate an mRNA having both messages. Translation formed a protein consisting of the total hormone polypeptide attached to the β-galactosidase fragment by a methionine residue. Cyanogen bromide cleaves peptide bonds at methionine residues. Treatment of the fusion protein with cyanogen bromide broke the peptide chain at the methionine and released the hormone. Once free, the polypeptide was able to fold properly and become active. Since production of the fusion protein was under the control of the lac operon, it could be easily regulated.