UNIVERSITI TUNKU ABDUL RAHMAN
FOUNDATION IN SCIENCE
SESSION 202210
TUTORIAL
BIOTECHNOLOGY II
FHSC1234 MODERN BIOLOGY
8
[Source: Final Examination September 2022]
Q1. Seven skeletons were discovered in a house in Pompeii, three of them were children.
It is believed they were inhabitants and workers within the house when Mount
Vesuvius erupted on 24 October 79 AD. Researchers were able to isolate DNA
samples from these skeletons and polymerase chain reaction (PCR) was conducted on
these samples followed by genetic fingerprinting to identify the skeletons. Figure 1
shows the results of the genetic fingerprinting of the three children and four adults.
Figure 1
(a) Explain why PCR was done on the DNA samples. (2 marks)
To produce many copies of the DNA sample to use for test (amplify as sample DNA obatain
is little)
(b) It was determined that the three children were siblings and shared the same
biological parents. Their mother is adult B. With justification, identify the
father. (2 marks)
1
Adult C as the first and last band of child 3 and last band of child 2 is same as Adult C
band. (whichever child bands that do not match the mother bands will
match the fathers so is C)
(c) During the PCR reaction, DNA was heated to 95°C and DNA primers,
nucleotides and a thermostable enzyme were added into the mixture.
(i) Explain why the DNA is heated to 95°C. (2 marks)
Denaturation to separate DNA double strand by breaking H bond
(ii) Describe what are DNA primers and state the purpose of using them in
PCR. (3 marks)
DNA primer is a short single strand nucleic acid which initiate the elongation of the
synthesis. Polymerase will add nucleotide base to the primer in 5’ to 3’ direction.
They are used in PCR to replicate the target sequences
(Short sequence of DNA that match target sequence / to match sample to be amplify, for taq
polymerase to carry out elongation/extension or add nucleotide to primer)
(iii) Name the thermostable enzyme. Explain your answer and state its
function. (3 marks)
Taq polymerase, as it is heat resistance which live in hot spring which remain functional in
heating process. It is used to add nucleotide to the primer on the daughter strand 5’ to
3’ direction.
[Source: Final Examination OCT 2018]
Q2. (a) Restriction enzymes are extensively used in molecular biology. Figure 2.1
shows the recognition sites of two enzymes, BamHI and BclI.
Figure 2.1
Figure 1.2 shows the sequence of a dsDNA molecule.
Figure 2.2
2
(i) Name the bond that is cut by restriction enzymes. (1 mark)
Phosphodiester bond
(ii) If the DNA segment in Figure 2.2 was cut with BamHI, state the
expected number of resulting DNA fragments produced and write the
sequences of these fragments. (3 marks)
1? fragment, 5’-A, T, T, G, A, G- 3’ 5’-GATCCTCTGATCACG-3’
(2 5’ ATTGAG 3’ 5’GATCCGTCTGATCACG 3’
3’ TAACTCCTAG 5’ 3’GCAGACTAGTGC 5’
(iii) “If the DNA segment was also cut with BclI, you may ligate the
restriction fragments produced with those cut with BamHI”. State
whether you agree with this statement, justify your answer. (3 marks)
No, in order to ligate, the nucleotide need to be cut with the same restriction
enzyme. And that the sequence cut by different enzyme is different.
(Yes, both produce complimentary sticky end which can form H bond with each other)
[Source: Final Examination Dec 2017]
Q3. Figure 3 shows the plasmid map of pBR322 vector.
Key:
Ampr: Ampicillin resistant gene
Tetr: Tetracycline resistant gene
Ori: Origin of replication
Figure 3
(a) State TWO (2) selective markers that can be found in this plasmid. (2 marks)
Ampr and Tetr
3
(b) Calculate the length of the DNA fragment(s) when the plasmid is hydrolysed
by the following enzyme(s).
(i) EcoR I only. (1 mark)
4363 bp
(ii) Hind III and BamH I. (2 marks)
346 bp, 4017 bp
Sam digested both Gene X and the plasmids using Pst I. He then “glued” the Gene X
into plasmids to form recombinant plasmids.
(i) Name the enzyme used to “glue” Gene X into plasmids. (1 mark)
DNA ligase
(ii) Name the covalent bond that results from the catalytic activity of the
enzyme mentioned in Q3. (c) (i). (1 mark)
Phosphodiester bond
(iii) Explain why Sam used the same restriction enzyme, Pst I, to digest
both Gene X and the plasmid. (2 marks)
To make sure the nucleotide base at the sticky end or the end is same
for both plasmid and DNA to be able to form bond
complementary. (complementary base pair so can H bond
complementary)
(iv) The success rate of getting recombinant plasmids was about 50%.
Briefly explain what happened during the insertion process that
contributed to this situation. (2 marks)
Not all bacteria is recombinant by taking in recombinant DNA. (reannealing/ recircularized
of plasmid or reannealing of target gene as they are complement to each other)
(c) Sam transformed E. coli bacterial cells with the recombinant plasmids from
Q3. (c) and plated the bacteria on agar. Three subsets of bacteria may grow: E.
coli with recombinant plasmids, E. coli with non-recombinant plasmids or
untransformed E. coli.
(i) State the colour(s) of the colonies formed if the agar were
supplemented with X-gal and tetracycline. Justify your answer.
(3 marks)
white colonies due to lack of laz G gene in plasmid so no beta galactosidase is not presence
so no digest of X gal
(ii) If the agar were supplemented with ampicillin, state whether E. coli
with recombinant plasmids would grow. Justify your answer.
4
(3 marks)
No as the sequence of ampicillin resistance gene is disrupt when pst1 cut through it as it is
within amp resistance gene. Hence no resistance to ampicillin so cell will die in presence of
ampicillin.
(iii) If the agar were supplemented with tetracycline and ampicillin, state
which subset(s) of bacteria would grow. Justify your answer.
(2 marks)
Non recombinant plasmid as it contain both resistance gene is not disrupted/ intact/ fully
functional
