Unit 4 SAC 1 - Gene Technology 2020

Section A: Multiple Choice Questions

Question 1
A specific restriction endonuclease, such as Eco R1,
A. joins pieces of DNA.
B. will produce three fragments when it cuts a linear eukaryotic allele three times.
C. can produce sticky ends or blunt ends depending on the cutting site it uses.
D. cuts at a specific recognition site.

Question 2
In preparing a recombinant plasmid for the insertion of a selected gene into a bacterium, it is most
important to
A. cut the plasmid with the same type of restriction enzyme that was used to cut the gene out
of the ‘donor’ DNA, so that complementary ‘blunt ends’ are produced.
B. produce complementary sticky ends both in the plasmid and on the ends of the inserted
‘foreign’ DNA
C. choose a plasmid which contains antibiotic resistance genes.
D. cut the plasmid using a restriction enzyme that produces ‘sticky ends’ which are
complementary to DNA ligase.

Use the following information to answer Questions 3 and 4.

Genetic engineers use restriction enzymes to cut DNA into smaller lengths. The recognition
sequences of several restriction enzymes are shown in the table below. The symbol * denotes the
restriction site (position of the cut).

Question 3
Consider a length of double-stranded DNA with the sequence

5’ T T A A G G A T T C A G G C C A C 3’
3’ A A T T C C T A A G T C C G G T G 5’

Adding EcoR1 and HaeIII to a solution containing one copy of this double-stranded DNA produces
A. three fragments of double-stranded DNA, each with a sticky end.
B. three fragments of single-stranded DNA, each with a sticky end.
C. three fragments of double-stranded DNA, each with blunt ends.
D. two fragments of double-stranded DNA, each with blunt ends.
Question 4
Now consider a different length of double-stranded DNA with the sequence

5’ C T T A A G C T A C C C A A T C A 3’
3’ G A A T T C G A T G G G T T A G T 5’

Which enzyme(s) will cut this piece of DNA?

A. EcoRI only
B. AluI only
C. AluI and HindIII only
D. AluI, HindIII and HaeIII only

Use the following information to answer Questions 5–7.

Bacteria can be transformed with an insulin gene and cultured to make insulin in commercial
quantities.
The steps taken to produce genetically engineered insulin are summarised below. The order of the
steps has been mixed up.

Q Both the human DNA
P
Culture transformed
Insert insulin gene into
bacteria and harvest
cut plasmid.
and purify the insulin
from the culture
R
and plasmid are
treated with the same
restriction enzyme to
produce identical sticky
ends

T U V
S
A human insulin gene is Add recombinant An engineered plasmid
DNA ligase
isolated from a cell and plasmids to bacteria containing certain
permanently bonds the
introns removed and heat to enhance restriction sites is
sticky ends.
uptake of plasmids prepared

Question 5
The correct sequence of steps when producing the insulin is
A. T, V, R, P, S, U, Q.
B. V, T, P, U, S, Q, R.
C. T, Q, V, R, P, U, S.
D. R, V, Q, T, P, S, U.

Question 6
The type of permanent bonds created at step S are
A. disulphide bridges.
B. peptide bonds.
C. hydrogen bonds.
D. phosphodiester bonds.
Question 7
To produce and market insulin, a human insulin gene used by one gene technology company may
have a different nucleotide sequence to the human insulin gene used by a second company.
This is because the DNA code is
A. contaminated by bacterial DNA.
B. universal.
C. mutated by heat shock.
D. redundant.

Question 8
Gel electrophoresis is a process used to separate DNA fragments whereby:
A. shorter fragments move through the pores in the gel more easily than longer fragments.
B. longer fragments move through the pores in the gel more easily than shorter fragments.
C. lighter fragments move through the pores in the gel more easily than heavier fragments.
D. heavier fragments move through the pores in the gel more easily than lighter fragments.

Question 9
Which of the following is a definition of transcription?
A. The synthesis of a polypeptide chain as mRNA is read by a ribosome.
B. The synthesis of RNA from a DNA template.
C. The synthesis of DNA from an RNA template.
D. The synthesis of mRNA from pre-mRNA

Question 10
Friedrich’s ataxia is an inherited disorder that causes progressive damage to the nervous system.
Friedrich’s ataxia is due to a mutation in the X25 gene that codes for the protein frataxin, a 210
amino acid protein. The onset of symptoms usually occurs between the ages of 5 and 15 years.
The mutation in this gene results in GAA triplet repeats. Normal alleles have 7 - 22 GAA repeats
whereas mutant alleles have 20 – 2000 GAA repeats in intron 1 of the frataxin gene. These repeats
interfere with transcription.
GAA codes for the amino acid leucine. Would this amino acid be added to the polypeptide chain in
multiple numbers in a patient suffering Friedrich’s ataxia?
A. Yes, as 20 – 2000 repeats of GAA occur in the genome of Friedrich’s ataxia sufferers.
B. No, as the multiple numbers of leucine molecules would be destroyed after gene regulation in
Friedrich’s ataxia sufferers.
C. Yes, as the multiple numbers of leucine molecules cannot be prevented with the destruction
of the regulatory gene.
D. No, as 20 – 2000 repeats of GAA occur in introns of Friedrich’s ataxia sufferers.
Question 11
The following diagram indicates the cutting sites of three different restriction enzymes on a
particular bacterial plasmid.

If the plasmid was incubated with the restriction enzyme Sal l, the number of pieces of DNA
obtained would be
A. two.
B. three.
C. four.
D. seven.

Question 12
A biotechnologist has various tools to use when manipulating DNA.
Which row from the following table correctly matches the tool with the element involved in the
manipulation of DNA?

Ligase Endonuclease Plasmid Polymerase

A. cutting pasting vector replicating
B. pasting cutting vector replicating
C. replicating cutting pasting vector
D. pasting vector replicating cutting
 Use the following information to answer Questions 13 and 14.

A paternity case was solved using genetic profiling. Gene loci from the mother, child and two
men were investigated to determine which of the two men was the father of the child. The
diagram below shows the results of the gel electrophoresis procedure.

Question 13
The smallest gene locus analysed on the gel is from
A. the mother.
B. the child.
C. man 1.
D. man 2.

Question 14
A suitable conclusion that could be made would be that
A. man 1 is the father because two of his gene loci are similar to two of the child’s gene loci.
B. neither man can be the father, because all gene loci bands between one of them and the
child would need to match for them to be the father.
C. man 2 is the father because none of his loci bands match those of the mother.
D. no conclusion can be made, because even though band patterns may match, the
nucleotide sequences may be different for each band.
Question 15
The diagram below represents a DNA molecule and the position of the recognition sites for the
restriction enzymes BamHI, EcoRI, HaeIII and SalI.

Also shown is a diagram of an electrophoresis gel in which the lanes R, S, T and U show the
separation of DNA segments resulting from digestion of the molecule with one of the restriction
enzymes.

Which of the following shows the correct match between the lane and the restriction enzyme used
to digest the DNA molecule?
 Section B: Short Answer Questions

Question 1
Haemophilia A is a genetically-caused bleeding disorder caused by a mutation in the F8 gene which
provides instructions for making a protein called coagulation factor VIII. Coagulation factors are a
group of related proteins that are essential for the formation of blood clots. After an injury, clots
protect the body by sealing off damaged blood vessels and preventing further blood loss.
Mutations in the F8 gene lead to the production of an abnormal version of coagulation factor VIII
protein. The altered or missing protein cannot participate effectively in the blood clotting process. As
a result, blood clots cannot form properly in response to injury. These problems with blood clotting
lead to continuous bleeding that can be difficult to control. The mutation may cause severe
haemophilia A by almost eliminating the activity of coagulation factor VIII.
It is now possible to genetically test people to see if they carry the faulty allele. This test uses PCR,
the restriction enzyme Sth134I and gel electrophoresis.
Sth134I is a restriction enzyme, sourced from the bacterium Streptococcus thermophilus, that
recognises the 4-base sequence in DNA,

C C G G
G G C C

and cuts it between the C and the G to produce

C C G G
G G C C

a. What term is used to describe the ends of the fragments produced by Sth134I? 1 mark
Sticky ends

Molecular studies have shown that the haemophilia A allele differs only by one base pair from the
normal allele. This base change occurs in a 4-base sequence that is recognised by the restriction
enzyme Sth134I. Two Sth134I sites are found within the normal allele, while only one Sth134I exists
within the haemophilia allele that is used in the genetic test.

108 bp 242 bp 78 bp

Normal allele

350 bp 78 bp

Haemophilia A allele

The PCR products are digested using Sth134I. The resulting fragments undergo gel electrophoresis.
b. How is the action of the Sth134I enzyme affected by the haemophilia A mutation? 2 marks
One restriction/recognition site is removed/destroyed in the haemophilia allele (1)
Hence, the normal allele is cut at two restrictions sites / Sth134I produces 3 fragments (0.5)
while the haemophilia allele is only cut at one restriction site / Sth134I produces 2
fragments (0.5)

The normal allele for the F8 gene is XH, while the haemophilia allele is denoted Xh.
c. Mark on the picture of the gel below the banding patterns you could expect to see for
someone who has each of the following genotypes. 2 marks
i. XH XH
ii. XH Xh

i. XH XH ii. XH Xh

_____ loading wells

+
Three bands for XH XH (0.5); four bands for XH Xh (0.5)
Relative locations of bands are correct when all seven bands are compared (1)

d. Restriction sites for EcoR1 exist at the same location on each side of the F8 gene, whether it is
a normal or haemophilia allele. Explain why these sites not used in the detection of
haemophilia? 2 marks
Both the normal and haemophilia alleles are of the same size (1)
Hence, both alleles would run together on the gel (0.5)
and detection of the faulty allele would be impossible (0.5)
Question 2
Cystic fibrosis (CF) is a recessive disorder affecting the chloride channels in cell membranes. It causes
the production of abnormally thick mucous, leading to the blockage of the pancreatic ducts,
intestines, and bronchi, often resulting in respiratory infection.
The faulty CF allele is three bases shorter in length than the normal allele.
Jo, who is currently pregnant, and Lee are parents of Betty and John. After already giving birth to a
CF child, Jo and Lee decided to have an amniocentesis procedure performed so that cells from the
unborn child could be tested for the presence of the CF allele.

b. i. Jo and Lee are considered to be carriers of cystic fibrosis. What does this mean?
1 mark

While Jo and Lee do not suffer CF / are normal (0.5) each carries a CF allele (0.5)

ii. Considering that each parent exhibits two bands in their DNA profiles, why does Betty
only display one band? 1 mark

Betty is homozygous / has two copies of the same allele (1)

iii. Will the unborn child suffer from cystic fibrosis? Explain. 1 mark

Yes (no mark)

The child has two copies of the shorter CF allele / is homozygous recessive (1)

iv. What is the chance that John could father CF children? Explain. 1 mark

It is impossible for John to father a CF child (0.5)

he only possesses normal alleles/is homozygous normal and can only pass on normal
alleles to any of his children (0.5)
Question 3 (VCAA 2017)
Scientists use recombinant bacterial plasmids as vectors to transform bacteria for a range of
purposes in research and biotechnology.
a. What is meant by the term ‘vector’ in the context given? 1 mark

Any method, often a plasmid or virus, that is used to transfer foreign DNA into a host cell

DNA can be inserted into bacterial plasmids to produce recombinant DNA.

The diagrams below show a bacterial plasmid in its original form and the recombinant plasmid after
a desired gene, gene X, which codes for human growth hormone, has been sliced into a particular
position. The plasmids are then incorporated into bacterial hosts.

KEY
Gene A: codes for resistance to the antibiotic Ampicillin
Gene B: codes for the production of a yellow fluorescent pigment
Gene X: codes for the production of human growth hormone

c. A gene technology company only desires surviving bacteria that have incorporated the
recombinant plasmid. Explain how gene A and gene B can be used to determine which
bacterial hosts have been successfully transformed for use by the company. 3 marks
i. an ampicillin-resistant gene A

When ampicillin is added, all bacteria which did not take up either of the plasmids will
be killed (1).

ii. a yellow pigment gene B

The incorporation of gene X into a plasmid destroys gene B. (1)

Hence, of the surviving bacteria in ampicillin, those which glow yellow are not desired
by the company and only the surviving bacteria which cannot glow yellow
(containing gene X) are collected for growth hormone production/produce growth
hormone (1)
A relevant reference to “growth hormone” is required (0.5)