PAST YEAR QUESTIONS 

PSPM PST 2003/2004 

6. a. Discuss the process of producing human insulin through DNA recombinant technology using ​Escherichia
coli​. [12 ​marks​]

-mRNA for insulin is extracted from pancreatic cells 

-used as a template 
-synthesis of cDNA by reverse transcriptase 
-and DNA polymerase 
 
Or 
 
-DNA containing the insulin gene is isolated 
-the DNA is spliced using restriction enzyme 
-to form DNA fragments (including the one containing the required gene) 
-with sticky ends 
 
-Vectpr/plasmid from ​Escherichia coli​ is isolated 
-The vector/plasmid is cut (with the same restriction enzyme)/ complementary ends 
-to produce linear plasmid with sticky ends 
-cDNA/DNA fragment inserted into vector/plasmid 
-with aid of DNA ligase 
-to form recombinant DNA plasmid 
-Recombinant DNA plasmid is transferred to ​Escherichia coli/​ / transformation 
-Screening is performed 
-For cloning 
-and amplification 
-Insulin gene will be expressed 
-Insulin is extracted from ​Escherichia coli 

PSPM PST 2004/2005 

6. a. Explain the importance of the following terms involved in gene cloning:

i) Vector
- Agent for carrying foreign DNA segment 
- Able to replicate / amplify / duplicate freely / autonomously 
- Able to express cloned gene 
- Possesses marker genes for screening 
- Possesses multiple cloning sites 
- Cloned gene can be recovered to cut / restrict both DNA strands 
- Eg: Plasmid / phage λ (bacteriophage) // cosmid 

ii) Palindrome
- The same base sequence in opposite directions 
- On double stranded DNA 
- The recognition site for restriction enzyme 
- To cut / restrict both DNA strands 
- Eg:  
 

 
 
 iii) Sticky ends
- Sticky ends are produced when the restriction enzyme produce staggered cut 
- i.e. hanging complementary single strand of DNA at the cut ends 
- DNA with ​complementary​ sticky ends are able to anneal / join 
- Eg:  
 

 
[10 ​marks​]

b. With the aid of a diagram, explain the steps involved in the production of a recombinant plasmid using ​E.
coli​ and a human gene which has been isolated. [10 ​marks​]

- ​E. coli​ is lysed 

- Plasmid is extracted 
- Plasmid is cut / restricted 
- With the ​same​ restriction enzyme (as used for isolating the human gene) 
- Plasmids open 
- Plasmids and human gene fragment bear complementary sticky ends 
- Human gene fragment is ​inserted​ into the plasmid 
- Joined / ligated by DNA ligase 
PSPM PST 2005/2006

6. a. Discuss the required characteristics of plasmid and host used in the recombinant DNA technology.
[8 ​marks​]
Characteristics of the plasmid 
-contains origin of replication 
-plasmid replicates independently in host cell 
-contain multiple cloning sites // unique restriction sites 
-allow more DNA fragments to be inserted 
-contain selectable marker / ampR / lacZ 
- resistant to antibiotic / β-galactosidase activity 
-allow the cells that contain the recombinant DNA to be readily identified / screened 
-ability to express // amplify the cloned genes 

Characteristics of the host cell 

-able to carry/accept/receive the recombinant DNA by transformation 
-able to maintain the recombinant DNA /plasmid from one generation to another 
-able to propagate easily for the amplification of the recombinant DNA 
-able to express the cloned genes 

PSPM PST 2006/2007 

3. FIGURE 3​ shows the various steps involved in DNA cloning.

 
FIGURE 3 

a. Name enzyme P and the enzyme involved to produce molecule R. [2 marks]

Enzyme P : Restriction//Eco R1//Sma I//Bam H1 
Enzyme involved to produce molecule R : (DNA) ligase 

b. Name molecule R. [1 ​mark]​

Recombinant plasmid//Recombinant DNA 

c. Name the processes Q, S and T. [3 ​marks​]

Q : insertion//ligation 
S : transformation 
T : Amplification 

d. Referring to FIGURE 3 what is the next important stage required to produce insulin by genetic
engineering. [1 mark]
Screening//Expression 
 e. State THREE benefits of insulin produced by genetic engineering. [3 ​marks]​
-The gene used is from human 
-Insulin prodicd similar to human insulin // same function as human insulin 
-Non allergic 
-Cheaper // can be produce in large amount 

PSPM PST 2007/2008 

6. a. What is meant by the term vector in DNA cloning? Describe its characteristics. [8 ​marks​]
Meaning : 
-Vector is small​ DNA molecule 
-act as an​ ​agent to ​ ​carries foreign DNA fragments /​ gene of interest. 
-​To (be introduced into) a host cell. 
-(derived from) a plasmid / bacteriophage / cosmids / YAC  
 
 
Characteristics : 
-Able to accept foreign DNA. 
-Able to replicate freely in the host cell / autonomous // to produce / amplify a similar DNA 
fragments. 
-Able to express the cloned gene. 
-Possess selectable marrker gene / resistance to antibiotics / LacZ activity. 
-They are easy to be selected. 
-They contain an origin of replication / Ori 
-They have two or more restriction sites (recognized by restriction enzymes) multiple cloning 
sites / unique restriction site. 

b. B​riefly describe the Polymerase Chain Reaction (PCR). [12 ​marks​]

-PCR is​ in-vitro process. 
-Involves a three step cycle 
-First step is ​denaturation /​ separation of double-stranded DNA. 
-By ​heating a high temperature / 94 - 98°C 
-Second step is​ ​annealing of the primer​ to the target region / sequence os single-stranded 
denatured DNA. 
-​By cooling ​/ lowering the t​emperature / 50 – 65°C 
-Third step is s​trand elongation / polymerization. 
-By​ primer extension. 
-At ​70 – 72 °C 
-Catalyzed by ​DNA polymerase​ / ​Taq​ DNA polymerase 
-Which is heat stable / resistance. 
-By adding the free nucleotide 
-In the 5’ to 3’ direction. 
-The cycle is repeated many times / 30 – 40 cycles ( 2​n​) 
Function of PCR: 
-To amplify identical DNA molecules. 
 
 
 
 
 
 
 PSPM PST 2008/2009  

2. FIGURE 2​ below shows part of the process involved in DNA cloning.

FIGURE 2 
a. State the characteristics that a plasmid must possess. [3 ​marks​]
- Antibiotic resistance ​gene​/marker gene/ selectable marker 
- Multiple cloning site (MCS)//unique restriction site//​able to accept foreign DNA/gene of interest 
- Origin of replication/ori/​can replicate independently 

b. Why the restriction enzyme ​Eco​RI is used to cut both the plasmid and foreign DNA? [1 ​mark]​
To produce​ same /identical/​compatible​//complementary ​sticky ends 

c. Name the enzyme needed for the process A. [1 ​mark]​

DNA ligase 
 
d. Name process B that introduced a recombinant plasmid into the host cell. [1 ​mark]​
Transformation 
 
e. Give the reasons why bacterial cells is suitable as a host. [3 ​marks​]
-Able to receive rDNA/r plasmid 
-Able to maintain the structure of rDNA 
-Able to amplify rDNA 
-Able to express cloned gene 

f. Name process ​C​ which is needed for ​identification​ of the transformed bacteria. [1 ​mark]​
Screening 
 
 
 
 
 
 
 
 
 
  
 
PSPM PST 2010/2011 

2. FIGURE 2​ shows the steps in gene technology.

FIGURE 2 

a. Identify A. [1 ​mark]​
mRNA/messenger RNA 

b. Name enzyme Y that catalyses the production of B. [1 ​mark​]

reverse transcriptase 
 
c. C is produced with the aid of DNA polymerase.
i) Name C. [1 ​mark]​
cDNA// double stranded DNA 
 
ii) What is the role of DNA polymerase in this process? [1 ​mark]​
Synthesis DNA from single stranded DNA 

iii) Give TWO applications of the copies of C. [2 ​marks​]

-make copies of DNA from mRNA​//m​ake copies of gene of interest 
-establish cDNA library 
 
d. i) Name process D. [1 ​mark]​
Amplification/ PCR 

ii) State ​TWO​ materials used in process D. [2 ​marks​]

-​ DNA Nucleotides 
-DNA polymerase/​Taq DNA polymerase 
-Primers 
-DNA template 

iii) State the most important characteristic of the enzyme involved in process D. [1 ​mark]​
Heat stable//heat resistance 

PSPM PST 2011/2012 

 
9. a. Discuss the process of cloning a​ human​ gene for ​protein production in bacteri​a. [12 ​marks​]
-Isolate RNA from human tissue 
-Using the reverse transcriptase enzyme 
-Form / produce the first DNA strand / single strand cDNA 
-Hydrolysis / degradation of the RNA molecule / strand 
-Synthesis of the second DNA strand // double stranded DNA/ form the complementary DNA 
(cDNA) 
-Catalysed by DNA polymerase 
-Ligation of adaptor DNA carrying a restriction site 
-Isolation of plasmid DNA (vector) from bacteria ​E. coli 
 -Cut both the plasmid DNA (vector) and cDNA (insert) with the same restriction enzyme 
-Insert cDNA / gene of interest into plasmid 
 
-Ligation of vector and insert using (T4 DNA) ligase 
-Produce recombinant DNA / plasmid 
-Transformation of recombinant DNA into host cell (​E. Coli​) 
-Bacteria amplification 
-Screening of recombinant colonies 
-Induce the expression of the cloned gene 

b. Describe the Polymerase Chain Reaction. [8 ​marks​]

-To amplify DNA 
-Through ​three​ steps of recycling reaction 
-Denaturation / to separate double stranded DNA 
-Annealing of primers to the (DNA) template 
-Primer extension to produce complementary DNA strand 
-Catalysed by (heat stable) DNA Polymerase (Taq Polymerase) 
-The cycle is repeated (many times) 
-Requires the ​four​ nucleotides (ATP, GTP, CTP, TTP) 
-Produce multiple / many copies of DNA/ amplify DNA 
-PCR is in-vitro process 

PSPM PST 2012/2013 

1. FIGURE 3​ shows steps involved in the ​cloning of human insulin gene.

FIGURE 3 

a. Name molecules A, B and C. [3 marks]

A: mRNA 
B: cDNA 
C:​ plasmid / vector​ // ​recombinant plasmid​ / recombinant DNA 

b. Name enzyme X and enzyme Y [2 marks]

X: Reverse transcriptase 
Y: DNA polymerase 

c. Why is the insulin gene obtained from A rather than the DNA? [1 mark]
 DNA contain introns // mRNA that copied from DNA does not contain any intron 

d. Name process D. [1 mark]

Transformation 

e. Why does the recombinant plasmid able to replicate autonomously inside the host? [1 mark]
It contain origin of replication / ori gene 

f. i) What is the importance of marker gene at C? [1 mark]

For screening / selection process 

ii) Give ONE example of marker gene. [1 mark]

​lacZ // antibiotic resistance gene // ampR // tetR 

5. a. Explain tools used in recombinant DNA technology [10 marks]

- Target DNA (gene) 
- DNA fragment that contain gene of interest to be cloned  
- Will be inserted into vector / plasmid 
- DNA cloning vector / plasmid 
- Vehicle / agent used in gene cloning // carry a foreign DNA fragment into the host cell 
- Restriction enzyme 
- Enzyme that recognize specific base sequence of DNA // cut / cleave the DNA at specific 
base sequence / restriction site 
- Modifying enzyme 
- Ligate the gene of interest and vector to form recombinant DNA 
- Host cell 
- Organism that receive recombinant DNA for cloning purpose 

b. Describe the steps involved in gene cloning [10 marks]

- Isolation of target DNA / gene and plasmid/ cloning vector 
- Cleave / cut the both DNA and plasmid by the same restriction enzymes 
- to have compatible sticky / blunt ends for successful combination 
- Target DNA / gene and plasmid / cloning vector joined / ligated by DNA ligase 
- to form a recombinant DNA 
- the recombinant DNA is transferred into host cell 
- by transformation process 
- recombinant plasmid / DNA / cloning vector is amplified in the host cell 
- followed by screening /selection process 
- to detect host cell containing recombinant DNA 
- positive cell colonies is cultured for further use 
- white colony contain recombinant DNA 
PSPM PDT 2012/2013 

4. a. List FOUR stages in the production of recombinant DNA. [4 marks]

Isolations of gene 
Cleave/ cut 
Insertion 
Transformation and amplification 

b. What is the function of electrophoresis gel? [1 mark]

To separate the DNA samples into different lenght. OOS 

c. State the role of restriction enzymes in recombinant DNA technology. [1 mark]

Recognize​ a specific sequences of bases on the DNA molecules and cut DNA at specific  
point/site/restriction site 

d. Give the term given to plants that are genetically modified [1 mark]
Trangenic plant 

e. Name TWO cloning vectors [2 marks]

Plasmid 
cosmid 

f. Give ONE example on how recombinant DNA technology can be used to protect the environment.
[1 mark]
Degradation of oil spill. 

8. a. Describe the characteristics of the cloning vector and host cell, ​Escherichia coli​ [12 marks]
Characteristic cloning vector 

- Contain origin of replication. 

- Plasmid replicates independently. 
- Contains multiple cloning site// unique restriction sites. 
- Allow much of DNA fragments to be inserted. 
- Contain selectable marker. 
- Resistant to antibiotic  
- Allow the cells that contain the recombinant DNA to be readily identified/ screened. 
- Ability to express // amplify cloned genes. 
 

Characteristic of host cell. 

- Able to carry/accept/receive the recombinant DNA by transformation. 

- Able to maintain the recombinant plasmid from one generation to other. 
- Able to propagate easily for the amplification of the recombinant DNA. 
- Able to express the cloned gene. 

b. Discuss the applications and ethics in the recombinant DNA technology in medicine [8 marks]

Application  

- Develop human insulin by using the bacteria as a host cell. 

- This hormone is responsible for controlling the glucose level in human. 
- Develop vaccine by cloning the gene used for protective antigen protein. 
- Develope human growth hormones. 
 - Responsible for growth, reproduction of the cells and regeneration in human as well 
as  
animals. 
- The disease of dwarfism is treated with this hormone. 
- Develope antibiotics 
- The chemical substances wich are used against bacteria infection. 
 

Ethics 

- highly cost in term of research, treatment, highly technical apparatus 

- human cloning is against the religion because it is an act to imitate God’s attribute 
that is  
creating human being 
- identity problem because cloning can lead identical problem of DNA 
- create gene castes 

PSPM PST 2013/2014 

8. a. What is gene cloning? Describe the steps involve in the process [12 marks]
 
- Gene cloning is a process of producing genetically identical copies of DNA. 
- Both donor / targeted gene / gene of interest ​and​ vector DNAs / plasmid is isolated. 
- The donor / targeted gene are amplified using PCR. 
- Targeted / donor gene ​and​ vector DNAs are cut. 
- Using the same /compatible restriction enzyme   / endonuclease to produce sticky  / blunt 
end. 
- Targeted / donor gene ​and​ vector DNAs are purified. 
- Targeted / donor gene ​and ​vector DNAs re ligated / inserted 
- Using (T4) DNA ligase 
- Forming recombinant DNA / plasmid 
- The  recombinant  DNA  /  plasmid  is  transformed  into  competent  host  /  cell.  // 
Transformation process. 
- Amplification process occurs. 
- (Blue white) screening for positive clones / recombinant plasmid. 
- Amplification of ​positive clones​. 

b. Describe the characteristics of cloning vector and host cell (bacteria) [8 marks]
Characteristics of cloning vector 

i. Can carry / accept foreign genes / DNA into bacteria / host cell. 
ii. Can be cloned / replicated independently inside host cell /contain ​ori​ / origin of replication. 
iii. Have unique / specific restriction sites / multiple cloning sites / MCS. 
iv. Has antibiotic of specific gene / selectable genetic marker / reported gene. 
v. Small in size (2.7 kb – 1.0 Mb). 
 
Characteristics of bacteria for cloning  
 
i. Can accept recombinant plasmid / DNA // cell competent 
ii. Antibiotic sensitive / resistance 
iii. Can clone itself / recombinant plasmid 
iv. Relatively larger than recombinant plasmid 
v. Able to maintain the structure of recombinant plasmid / DNA (from generation to generation) 
vi. Able to amplify / express the gene from recombinant plasmid / DNA. 
  

PSPM PST 2014/2015 

 
3. FIGURE 3 shows a construction of recombinant bacteria capable of degrading oil.

a. Identify steps P, Q, and R. [3 marks]

P : Gene inserted into plasmid / Insertion // Ligation 
Q : Transformation // Plasmid is transferred into bacterial cell 
R : Screening // amplification // propagation 

b. (i) Explain briefly step P. [3 marks]

Both plasmid ​and​ gene of interest / targeted gene/ DNA is cut / cleave / digest with ​same​ / 
compatible​ restriction enzyme. 
Allow both DNA to ligate / join. 
Using DNA ligase. 
 

(ii) State TWO the characteristic of cloning vector used in step P. [2 marks]
Able to accept / receive foreign DNA / gene of interest // presence of MSC 
Able to replicate freely / presence of ​ori
Having selectable marker gene // antibiotic resistant gene / ampicillin resistant gene. 
Having reporter gene. 

c. Give TWO characteristics of the recombinant bacteria in step R? [2 marks]

Resistant to antibiotic // ability to grow in antibiotic plate 
Able to form white colony 
Able to express foreign gene 
Able to maintain the structure of recombinant DNA 
Able to amplify gene of interest 
PSPM PDT 2013/2014 

8 (a) D​escribe the restriction enzyme​ and e​xplain how it cuts ​the DNA to form recombinant DNA [10 marks]

-Also known as restriction endonucleases 

-Usually isolated from bacterial cell 
-Eg: EcoRI isolated from ​Escherichia coli 
- To cut/cleave the DNA into fragments 
-by recognize and cleave at the specific short nucleotide sequences (restriction site) on DNA 
molecule 
- Eg: EcoRI restriction site is palindromic sequence of –GAATTC- , cut between G and A nucleotide 
- Eg: SmaI restriction site is palindromic sequence of -CCCGGG- , cut between C and G nucleotide 
- Restriction enzyme cut the sugar-phosphate backbones by breaking the phosphodiester bonds 
between nucleotide 
-Different restriction enzyme has different restriction site 
- Some restriction enzyme (such as EcoRI) leaves a staggered cut with single sticky ends.  
- Some restriction enzyme (such as SmaI) makes a straight cut across both strands at a single point, 
leaving blunt ends. 
- Both foreign DNA contain target gene and cloning vector (such as plasmid) need to be cut / cleave 
using the ​same restriction enzyme 
- so that ​compatible ends​ (either sticky or blunt) produce 
- Thus, both fragment DNA and opened plasmid able to ligate / join together by DNA ligase, by 
forming phosphodiester bond 
- to produce recombinant DNA / recombinant plasmid 

(b) Define cloning vector and explain THREE of its common type [10 marks]
- Agent that carries the foreign DNA fragments contain target gene to be introduced into a host cell 
- Type of DNA cloning vector are plasmid, bacteriophages, cosmids and yeast artificial chromosome 
(YAC) 
 
Plasmid: 
-a structure in bacterial cells that can replicate independently 
-small circular rings chromosome double stranded DNA that carry specific genes  
- Eg; gene for resistance to antibiotics (such as ampR), lacZ gene and origin of replication (ori) 
- Foreign fragment DNA will be inserted to the multiple cloning site (MCS or polylinker) within lacZ 
gene 
-can carry about 10-15 kilo base pairs 
- Host cell that receive this kind of recombinant plasmid can be screen / selected easily due to 
selectable genetic markers in plasmid 
- Eg: engineered plasmid pUC18 
 
Bacteriophages: 
- a type of virus that had been disable so that they do not cause disease in the host cells to which 
they are introduced 
- Eg: bacteriophage lambda (​λ2001)
- Foreign DNA can be packaged in phage particles, which can be used to infect suitable host cells 
- can carry about 15 kilo base pairs
 
Cosmids: 
- a hybrid cloning vector of plasmid and cos gene from lambda bacteriophage 
- also contain drug resistant, marker genes and other plasmid genes 
- can incorporate large DNA fragments than plasmid or phage 
 - suitable to clone large mammalian genes or multiple fragments DNA 
-can carry about 44 kilo base pairs 
-Eg: sCOS-1 
 
Yeast artificial chromosome (YAC) 
- modified plasmid used for cloning large segments of DNA in yeast cells 
- can carry a larger DNA fragments / an entire gene of DNA 
- can carry about 500 kilo base pairs  
 
PSPM PDT 2014/2015 

4. FIGURE 4 shows the schematic diagram of gene cloning.

(a) Labelled the structure X, Y and Z. [3 marks]

X : plasmid / cloning vector 
Y : target DNA / target gene / gene of interest / foreign gene / DNA fragment 
Z : recombinant DNA / plasmid // rDNA 

(b) State the function of X and Y [2 marks]

X : to carry target DNA / gene of interest / foreign gene / DNA fragment into the host cell 
Y : carry / contain gene of interest to be cloned 

(c) Name step I, II and III [3 marks]

I : cutting / cleave (of donor DNA and plasmid DNA with the same restriction enzyme) 
II : insertion (of DNA fragments into plasmid DNA) 
III : transformation (of recombinant DNA into the bacterial host cell) 

(d) (i) Name the enzyme used in step I [1 mark]

​Restriction enzyme / restriction endonuclease / ​Eco​RI / ​BamH
​ I  

(ii) What is the function of enzyme used in 4 (d) (i)? [1 mark]

​To cut / cleave the DNA at particular / palindromic / specific base sequences 
PSPM PDT 2015/2016 
 
2. FIGURE 2 ​shows the process of gene cloning.

 
 
i. What is the type of enzyme ​A​ and ​B​? [1 mark]
Restriction enzyme 

ii. How to confirm that the plasmid and target gene are fully digested with enzyme ​A​ and ​B​? [1 mark]
Separation using electrophoresis// compatible sticky end/blunt ends are formed. 
 
iii. Identify the process ​P​ and name the enzyme involved. [2 marks]
Process: Ligation//Insertion 
Enzyme: DNA Ligase. 

iv. Suggest the method used in ​Q​. [1 mark]

Calcium chloride (CaCl​2​) heat shock method//Electroporation. 

v. Name the bacteria that is commonly used as a host cell in gene cloning. [1 mark]
Escherichia​ ​coli​/ ​E​.​coli 

vi. State the method used to convert mRNA to DNA and name the enzyme involved.
Method: Reverse Transcription  
Enzyme: Reverse transcriptase 
[ 2 marks]
vii. Suggest ​TWO ​cloning vectors that are suitable to clone DNA fragment bigger than 20 kilobases.
Cosmid 
 Bacteriophage 
Yeast Artificial Chromosome (YAC) [2 marks]

6. a) Describe the basic principles of polymerase chain reaction. [12 marks]

i. Denaturation  
ii. Heating briefly/ the temperature is increased to 90-95​0​C 
iii. To separate two DNA strands/dsDNA 
iv. By breaking the hydrogen bonds between complementary bases. 
 
v. Annealing 
vi. By cooling/ lowering the temperature 50-65​0​C 
vii. To allow primers bind to the single stranded DNA template. 
viii. By formation of hydrogen bonds. 
 
ix. Extension/elongation 
x. The temperature is increased to 72​o​C for ​Taq​ polymerase 
xi. The ​Taq​ polymerase/ DNA polymerase synthesizes the new DNA strand complementary to the DNA 
template strand 
xii. In ​5’ to 3’ direction 
xiii. By adding DNA nucleotides/dNTPs 
xiv. The steps are repeated to produce millions of copies of the target DNA fragment. 
 
b) Explain the properties of plasmids as a cloning vector. [8 marks].
i. Small 
ii. Easy to insert into bacteria//allows the insertion of foreign DNA 
iii. Has selectable markers such as ​amp​R​/ ​lac​ Z gene// resistance to ampicillin 
iv. Allow bacterial cell containing plasmid to be isolated. 
v. Has unique restriction sites// has multiple cloning sites 
vi. Allows flexibility for cloning 
vii. Has the origin of replication 
viii. Allows plasmid to replicate within the host// rapid replication in a host cell// able to replicate freely 
in the host cell. 
ix. Gives higher yield. 
 

PSPM PST 2015/2016

8. a. Explain how a transgenic plant is produced using recombinant DNA technology. [12 marks]
i. Isolate mRNA of gene of interest 
ii. Example: resistant to herbicide/ pest/ diseases/ enhance taste/extend shelf life 
iii. Using reverse transcriptase 
iv. To produce cDNA 
v. Amplify the cDNA using PCR 
vi. Gene of interest/cDNA and Ti plasmid are cut 
vii. With the same/compatible restriction enzymes 
viii. Gene of interest is insert into plasmid/ vector 
ix. Using DNA ligase 
x. Produce Ti plasmid recombinant 
xi. Transform/ introduce into host cell eg: plant cell 
xii. Using soil bacterium/ ​Agrobacterium​ ​tumefaciens​/ gene gun/ electroporation 
xiii. Integrate with the host cell’s chromosome 
xiv. The transgenic plant will grow expressing the desired phenotype. 

b. Define cloning vector and host cell. Describe the characteristics of plasmids as cloning vectors and
characteristics of host cells. [8 marks]
i. Cloning vector is a DNA molecule that carry foreign DNA into a host cell and replicate them 
 ii. Host cell is bacterium/ animal/ plant cell that can serve as the living host for 
replication/amplification/ expression of recombinant DNA. 
 
Plasmids as cloning vector: 
iii. Able to accept/ carry foreign gene 
iv. Must have multiple cloning site/ MCS/ specific restriction site for cloning  
v. Able to replicate freely in the host cell/ have origin of replication ( origin of replication initiation-ori) 
vi. Must have selectable genetic marker/ antibiotic marker/ ampicillin resistance 
 
Host cell: 
vii. Able to receive recombinant DNA through transformation 
viii. Able to maintain the structure of recombinant DNA from one generation to the next 
ix. Able to amplify/ replicate/ multiply the gene product from the recombinant DNA 
x. Able to express the gene of interest. 

PSPM PST 2016/2017

3 (a) give two examples of genetically modified organism used in agriculture. [ 2 marks]
(Transgenic) soybean (resistant to herbicides) / tomato/potato/paddy/corn
/oil palm/cotton/banana/sugar beet/canola/papaya
(Transgenic) salmon/goat/cow (Any 2)

(b) Figure 3 shows the productions of human insulin using DNA recombinant technology.

(I) give two advantages of using complementary DNA (cDNA) in gene cloning. [ 2marks]
Consist of coding region of DNA / Code for specific target gene //
Consist of exon only
Suitable for (prokaryotic) host cell // intron is no longer exist //
(prokaryote) host cell can express the gene

(ii) Name molecule Q. [1mark]

Recombinant DNA / plasmid

(iii) Name an organism that can be used as a host in gene cloning. [1mark]
Escherichia coli​ / ​E.coli​ / ​E​.​coli​ / ​Escherichia​ ​coli
 (iV) Name the enzyme that joins the plasmid vector with the human cDNA. [1mark]
(DNA) ligase

(c) State two advantages of producing insulin through genetic engineering. [2marks]
Can be produced in large amounts // rapid production
Compatible to human insulin / no adverse reaction / no rejections / no side effects /non
allergic
Cheap / low cost / cost efficient
Halal / Syariah compliance

(d) Name another method that can be used to produce many copies of gene. [ 1mark]
Polymerase Chain Reaction / PCR

 
PSPM PDT 2016/2017 
 
2  Figure 2.1 shows the steps of gene cloning.

(a) Name the tools that are used in: [2 marks]

Step 1: restriction enzyme 
Step 3: Modifying enzyme /DNA ligase 
 
(b)  State the characteristic of the tool in step 2. [3marks] 
Specific to the restriction site 
Able to break phosphodiester bond 
Palindromic sequence 
Produce sticky end/blunt end 
 
(c)  Name the organism that is commonly used as a host cell. [1mark] 
E.coli ​/bacteria 
 
 (d)  Give two characteristic of the host cell in 2(c). [2marks] 
Receive DNA recombinant through the ​transformation ​process 
Maintain the structure of the recombinant DNA from one generationto another. 
Amplified /propagate/ multiply the gene produce from recombinant DNA. 
 
 
 
 
(e)  Figure 2.2 shows Plasmid M.

Gene of interest is inserted in between ampicillin gene. The recombinant DNA molecule is then
transformed into a host cell. Will the host cell survive in a medium containing ampicillin? Explain
briefly. [2marks]

No/ cannot survive. 

Because ampicillin gene/amp​R​ is distrupted/inactivated 
 
7 (a) Explain how human growth hormone can be produced in bacteria using recombinant DNA
technology. [13 marks]
 
● mRNA is isolated from (pituitary glands) of human. 
● mRNA is exposed to reversed transcriptase enzyme. 
● To produce complementary DNA(cDNA). 
● Both ends of cDNA are cut with specific restriction enzyme. 
● The plasmid (vector) is cut using the same restriction enzyme. 
● Producing  molecules  with  complementary single-stranded end/sticky end, blunt 
end. 
● Desired gene/gene of interest is inserted into plasmid at sticky end. 
● DNA ligase is used to form covalent bonds at junctions/linking fragment / both 
molecule//using DNA ligase. 
● Forming recombinant DNA/plasmid molecule. 
● Transfer/transform recombinant DNA molecule into specific bacteria. 
● Recombinant  DNA  molecules/  plasmid  multiply  in  bacteria//the desired gene is 
clone used  plasmid(from bacteria). 
● The  bacterial  cell  processes  turn  on the gene for human growth hormone//the 
bacteria cell  start to express human growth hormone gene. 
● The  growth  hormone  is  produce  in  the  cell//when  bacteria  cells reproduce by 
dividing, the  human growth hormone gene is also reproduce. 
● Human growth hormone molecule produce in bacteria are ( gathered) and purified. 
 
 
 
 (b) A forensic chemist realizes that he only has a tiny amount of tissue recovered from a crime scene.
Suggest and describe the technique he has to carry out in order to produce a large amount of DNA
for further analysis. [7 marks]

● The technique is Polymerase chain reaction/PCR. 

● DNA is extracted/ purified from the tissue. 
● Denaturation //DNA is denatured /separate into single strands by heating. 
● Temperature 94C - 98C . 
● Anneling//Primers are attached to primer-binding site /3’ end on each DNA strand. 
● Temperature 50C - 60C. 
● Extension//each DNA strand acts as a template for DNA synthesis. 
● Temperature 70C- 72C. 
● Catslysed by ​taq​ polymerase/PCR enzyme. 
● Number of DNA molecules doubles// will be propagated / multiply . 
● The DNA is heated again to repeat the cycle. 
● Cycle is repeated ( for 28 to 33 time) 
 
 
PSPM DB025 2017/2018 

3. Figure 2.1 shows the process of produsing recombinant DNA molecule.

(a) Name the bond that is digested (cut) by the restriction enzyme is step A. [1M]
​Phosphodiester bond

(b) Identify part B. [1M]

Sticky ends

(c) Name the enzyme that is involved in step C. [1M]

(DNA) ligase

(d) Give one example of plasmid. [1M]

pUC18 // Ti plasmid

(e) Give three characteristic of plasmid as a cloning vector. [3M]

accept foreign DNA at multiple cloning sites
able to replicate / amplify freely / independently in host cell
 possess selectable genetic marker / antibiotic resistance gene / ampR / ampicillin
resistance gene / possess lacZ

(f) Name a technique that is used to ensure a plasmid contains foreign DNA. [1M]
Blue-white screening / blue / white screening / lacZ selection

(g) Figure 2.2 is DNA sequence of a gene segment .figure 2.3 shows a restriction site for
enzyme SmaI.

Show how enzyme S​ma​I cuts the gene segment to produce blunt ends. [2M]

7. (a) Danial plans to grow transgenic plants which are resistance to insect and tumour growth
as indicators that these plants are genetically modified. Describe how to produce these
type of plant. [13M]
i. Isolate mRNA from a target cells / plant resistant to insect
ii. mRNA exposed to enzyme reverse transcriptase
ii. To produce complementary DNA / cDNA (which is double stranded)
iii. Cut / digest cDNA with restriction enzyme
iv. Isolate Ti plasmid from (bacterium) Agrobacterium (sp.)
v. Cut / digest Ti plasmid / vector (at one point) using the same restriction enzyme
vi. Produces molecules with complementary / compatible (sticky/blunt) ends
vii. cDNA / gene of interest inserted into the (middle of) T DNA segment
viii. DNA ligase to forms covalent / phosphodiester bonds at junctions // linking DNA
fragments
ix. Mix desired gene with cut plasmid to form recombinant DNA / rDNA / recombinant
plasmid molecule
x. Introduce recombinant DNA / rDNA / recombinant plasmid molecule into (bacterium)
Agrobacterium (sp.)
xi. Bacterium / Agrobacterium (sp.) exposed / introduced to plant cell
 xii. Once plasmid taken up, the desired gene is integrated into the host // plant cell’s
chromosomes;
xiii. Plant will grow tumor indicating it has the desired gene

(b) Describe the characteristic of restriction enzyme and give One example. [7M]
i. Restriction enzymes ​also known as restriction endonuclease
ii. ​Extracted from bacteria
iii. C​ut / cleave / digest double stranded DNA / phosphodiester bond
iv​. Recognise specific DNA sequence / restriction site
vi. C​ut / cleave / digest DNA at specific base within the sequence / recognition sites
vii. Recognition sites contain palindromes / palindromic / same sequence of bases
that can be read from both opposite direction
viii. Restric​tion enzyme produce staggered cuts / sticky ends or straight cut / blunt
end
ix. Example:​ EcoR ​ I / ​Bam​H1 / ​Sma​I

 
PSPM SB016 2017/2018 
 
9. (a) How is bacteria genetically engineered to produced human Insulin? [ 7M]
- ​mRNA for insulin isolatedfrom human pancreas 
- mRNA is used as a template //mRNA undergo reverse trancription  
- by reverse transcriptase  
- to produce single stranded DNA/cDNA 
- DNA polymerase is added to synthesize the second strand of complementary DNA/ double 
stranded cDNA. 
- cDNA a​nd​ plasmid are cut with same restriction enzyme/ endonuclease 
- to produce complementary sticky/ blunt end/ compatible end 
- the cDNA is inserted into the plasmid ​and​ join using DNA ligase 
- forming recombinant DNA / plasmid 
- transformed ​and​ amplification of bacteria/ host cell 
- blue white screening of the recombinant bacteria. 

(b) What are the differences between a plasmid and chromosome? [8 M]

-
plasmid  chromosome 
Circular DNA  Iinear ​or​ c​ ircular DNA 
Found in bacteria cells/prokaryote/yeast/Archaea  In prokaryotes​ or ​eukaryotes 
Facilitate conjugation  Does not facilitate conjugation 
Has  few  gene/  does  not  carry  vital  gene  for  Has many gene/ carry vital gene for cell 
cell/cary  selectable  genetic  marker/  antibiotic 
resistant gene 
Not needed for cell growth   Needed for cell growth 
Does not involve in heredity  Involve in heredity 
Not associate with protein  Associated with protein 
Can replicate independently in host cell  Replicate during cell division/ binary fission 
Has one origin of replication/​ ori  Has many origin of replication 
Use as cloning vector  Not use as cloning vector 

(c ) Give the definition for restriction enzyme and explain how these enzyme involved in
recombinant DNA technology. [5M]
 
- an enzyme that cut / cleave DNA at a restriction site/ recognition site 
- it cuts both vector/plasmid ​and​ of interest / target DNA 
- make staggered cut/ straight cut 
- to produce sticky end / blunt end/ compatible end 
 - producing free 5’ end ​and​ 3’ end 
- allow for DNA Ligase to ligate 
- producing recombinant DNA/ plasmid 
 
 
 
 
 
 
 
PSPM S2S 2018/2019

1. (a) (i) Describe the functions of restriction in gene cloning.

[2 M]

- produce sticky /complementary ​or ​blunt ends

- recognise ​and​ cut/ cleave DNA at specific / restriction site
- it break the phosphodiester bond/linkage

(i) What are the characteristic of a good host cell to be used in recombinant DNA
technology? - able to receive /accept cloning vector/ recombinant DNA/
recombinant plasmid
- able to maintain the cloning vector / recombinant DNA
- able to express the gene of interest within cloning vector
- able to amplify/proliferate the cloning vector/recombinant DNA
[2M]

(ii) Name the enzyme used to join DNA fragments and state how it works. [2M]

- DNA ligase
- (links/join DNA fragments) by forming phosphodiester bond/ linkage

(b) (I) Describe how recombinant plasmid are formed. [4M]

- isolate donor/target DNA /gene of interest ​and​ plasmid

- cut both /donor DNA and plasmid using the same restriction enzyme.
- to produce compatible end/ complementary end.
- insertion of DNA fragment into plasmid using DNA ligase// Mix both fragment
together using DNA ligase

(iii) Explain the application of blue/white screening in selecting recombinant plasmid [3M]

- transformed bacteria is plated/ cultured on a medium with antibiotic/ampicillin ​and

X-gal.
- bacteria with non-recombinant plasmid have functionl ​lacZ​ form blue colonies//
- bacteria with non-recombinant plasmid able to produce ​β​- galactosidase form blue
colonies.//
- bacteria with non-recombinant plasmid able to hydrolyze X-gal, form blue colonies
- Bacteria with recombinant plasmid have non-functional ​lac​Z form white colonies.//
- Bacteria with recombinant plasmid unable to produce ​β​-galactosidase form white
colonies//
- Bacteria with recombinant plasmid unable to hydrolyze X-gal form white colonies.

( c) Describe the steps in the production of insulin cDNA. [7M]

-isolate insulin mRNA

- use reverse transcriptase
- use mRNA as a template
- produce /synthesize single strand cDNA
- mRNA is degraded/ hydrolyze
- single strand cDNA act as a template
- to produce second strand of cDNA // to produce double strand cDNA
- by using DNA polymerase