Biology Students Resources SB015

BIOLOGY
SCORE

CHAPTER 8: RECOMBINANT DNA TECHNOLOGY

SUBTOPIC : 8.1 Recombinant DNA Technology

LEARNING OUTCOMES : a) Define recombinant DNA technology.
b) Define and explain the tools used in recombinant DNA technology, target
DNA, restriction enzymes, DNA cloning vector, host cell and modifying
enzymes.
(c) Explain restriction enzyme and examples of enzymes that produce sticky ends.
(EcoRI: G AATTC) and blunt ends (SmaI : CCC GGG)
(d) Explain the characteristics of plasmid as cloning and expression vector
(e) Explain the characteristic of E.coli as host cell and its characteristics
(f) Explain modifying enzyme and its function; (i) DNA ligase for DNA ligation
(ii) Taq polymerase for DNA
amplification using PCR.
.
MAIN IDEAS
EXPLANATION NOTES
/KEY POINT
Recombinant It’s a technology that forms a new combination of DNA when
DNA segments of DNA from two different sources (often different
technology species) in vitro.
• Enable scientists to obtain copies of specific DNA segment
for the purpose of studying it.
Purpose
• Modifying the DNA of an organism to produces new genes
with new traits.

1. Target DNA (Genes of interests).

It’s a fragment of chromosomal to be cloned. An enzyme
must be used to cleave fragments that containing genes of
interests.

2. Restriction Enzymes / Restriction of endonucleases.

Tools Used in This enzyme is extracted from a specific bacteria, for example
Cloning
E.coli. Naturally it is used to cut/splice viral DNA into small
fragments at specific base sequence/ restriction sites. Most of
the base are palindromic. Palindromic is a base sequence of one
strand reads the same as its complement strand in opposite
direction.

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5’ 3’
G A A T T C
C T T A A G
3’ 5’

3. DNA cloning vector

A small pieces of DNA which a foreign DNA fragment can
be inserted. For examples, plasmid, bacteriophage, cosmid
and YACs. Every vector must have these characteristics:
1. Able to accept foreign DNA in multiple cloning sites
(MCS)
2. Able to replicate freely in host cell.
Present of origin of replication initiation -ori gene
3. Possess selectable genetic marker
a. resistance to antibiotic.
eg: ampR, tetR, kanR
b. lacZ gene
encode for B- galactosidase

amp R

lacZ

4. Host Cell
A host cell is a cell that has been introduced
with DNA (or RNA), such as a bacterial cell acting as a host
cell for the DNA isolated from a bacteriophage. This cell is
utilized from the DNA cloning to accept, maintain and allow
the replication of cloning vector. This cell needs to have
these characteristics:
1. Able to receive DNA through the transformation
processes.
2. Able to maintain the structure of DNA recombinant
from one generation to other.
3. Able to amplify the gene product from the DNA
recombinant
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5. Modifying Enzymes
This is an enzyme used in the modification of DNA. For
example, DNA ligase. This enzyme catalyzed the formation of
phosphodiester bond between adjacent nucleotides in DNA.
Another example is, Taq polymerase. This is a DNA
polymerase isolated from thermophilic bacterium called
Thermus aquaticus. It catalyzes the addition of nucleotides to
DNA sequences. This enzyme is able to withstand high
temperature thus, it won’t be denatured.

Each restriction enzyme is very specific, recognizing a

particular short DNA sequence (restriction site), and cutting
both DNA strands at precise points within this restriction site.
(Campbell, 2011). Most restriction enzyme make staggered cut
in two strands, forming sticky end. For example, EcoRI. But
some enzyme could also cut straight across both strand, forming
blunt end. For example, SmaI. The table below shows the
Restriction restriction site that specifies the restriction enzyme.
enzyme,
examples of
enzymes that Enzymes Restriction Sites
produce sticky
ends. EcoRI 5’-GAATTC-3’
3’-CTTAAG-5’

BamHI 5’-GGATCC-3’
3’-CCTAGG-5’
SmaI 5’-GGGCCC-3’
3’-CCCGGG-5’

The list and The original plasmid is called a cloning vector, defined as a
explanation the DNA molecule that can carry foreign DNA into a host cell
types of cloning and replicate there. These are the examples of cloning
vectors.
vectors.

1) Plasmid
It is a small ring-shape DNA in bacteria (only a small
number of genes). It has the form of double stranded
circular DNA. However, it is not part of the bacteria’s
chromosome. But, it can self – replicating. For example,
pUC18

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Bacterial DNA Plasmid

2) Bacteriophage
Bacteriophage is a virus that infected bacteria. The genetic
information is in a linear form. It carries a larger DNA
inserts capacity then the bacteria. For example, λ2001

Genetic information

3) Cosmid
Cosmids can be used to build genomic libraries. Cosmids
can contain 37 to 52 kb of DNA, limits based on the
normal bacteriophage packaging size. They can replicate
as plasmids if they have a suitable origin of replication
(ori): for example SV40 ori in mammalian cells, ColE1 ori
for double-stranded DNA replication, They frequently
also contain a gene for selection such as antibiotic
resistance, so that the transformed cells can be identified
by plating on a medium containing the antibiotic. (Hybrids
of plasmids and bacteriophage lambda DNA). Other
example is SCOS-1

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4) YACs
Yeast artificial chromosomes (YACs) are genetically
engineered chromosomes derived from the DNA of the
yeast, Saccharomyces cerevisiae, which is then ligated into
a bacterial plasmid. By inserting large fragments of DNA,
from 100–1000 kb, the inserted sequences can be cloned
and physically mapped using a process called chromosome
walking. This is the process that was initially used for
the Human Genome Project, however due to stability issues,
YACs were abandoned for the use of Bacterial artificial
chromosomes (BAC). This chromosome is a double
stranded circular DNA. Other words, it is a plasmid with
yeast chromosome.
• Eg. pYAC

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BIOLOGY
SCORE

CHAPTER 8: RECOMBINANT DNA TECHNOLOGY

SUBTOPIC : 8.2 Methods in Gene Cloning
LEARNING OUTCOMES : a) Overview using diagram to show the steps in gene cloning by using plasmid
b) Describe the steps in gene cloning by using plasmid as the vector
(i) isolation of gene
(ii) cleave/cut
(iii) insertion
(iv) transformation and amplification
(v) screening (blue/white screening)

MAIN IDEAS
EXPLANATION NOTES
/KEY POINT

Cloning is the process of producing genetically identical individuals

Cloning
of an organism either naturally or artificially.

Is a process that produce many copies of a gene of interest by

making many identical copies of a gene by inserting the gene into a
living host cell (bacteria). These copies can be used in sequencing
the gene.
Gene Cloning
• 2 methods to copies the DNA fragment:
a. Cloning
b. Polymerase Chain Reaction (PCR)

1. Isolation of gene and vector (plasmid) from source (different

organisms).
• Isolation of plasmid (as a vector) from bacteria.
Steps in Gene
Cloning (Refer
to Campbell,
2011, (9th Ed.),
Pg. 445)

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• Isolation of target DNA or gene of interest from sources

Eg: Human cells, Plant cells, Animal cells

2. Cleave or cut of DNA/gene of interest and plasmid by using the

same restriction enzyme
• The target DNA and plasmid are cut at palindromic
sequence (restriction site) with the same restriction enzyme.
Single cut within the lacZ gene, caused the plasmid to open
and the disruption of the gene. However, many cuts within
the target DNA produced thousands of fragments with
different sizes.

3. Insertion of gene of interest into plasmid (vector).

• Mix the cut plasmid and DNA fragments allowing base pair
between their complementary sticky ends by forming
hydrogen bonds. DNA ligase is added to seal them together
at the sugar phosphate backbones of the fragments whose
sticky ends have base-paired with phosphodiester bond.
Thus, forming a recombinant DNA.

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• BUT, not all plasmid will join with the target DNA. This
caused two types of recombinant DNA:

 Non recombinant plasmid

 Recombinant plasmid

Inserted gene of
interest

4. Transformation of recombinant DNA into host cells (bacteria)

and amplification.
– Host cells take up recombinant DNA after being
introduced to it. BUT, not all host cells will take in
the recombinant DNA. Only a small proportion of
bacteria will be transformed.

• There will be THREE conditions:

1. Transformed with non-recombinant
plasmid
2. Transformed with recombinant
plasmid
3. Not transformed

Then, they are mixed in a medium containing calcium chloride. This

solution caused the bacterial cell wall to become permeable. The
host cells reproduce by binary fission and amplify the target DNA.

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5. Screening
The mixed bacteria are plate on nutrient-containing agar
medium supplemented with ampicillin and X-gal, a molecule
resembling lactose.
– Purpose: To identify bacterial colonies that carry
gene of interest / target DNA = Blue-white
screening.

• A plasmid vector contains two genes encoding for :

– Resistance to antibiotic (ampR)
– Enzyme β-galactosidase (lacZ)
–
• When cultured in medium containing ampicilin and X-gal:
Bacteria without plasmid fail to grow because of
none ampR gene – not resistance to antibiotic

• Bacteria with plasmid live but with TWO conditions:

– Contain non-recombinant plasmids.
• lacZ gene that encodes for β-galactosidase is
functioning
• Hydrolyzed X-gal  BLUE colonies

– Contain recombinant plasmids.

• lacZ gene that encodes for β-galactosidase is
disrupted.
• X-gal not hydrolyzed  WHITE colonies.

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BIOLOGY
SCORE

CHAPTER 8 : RECOMBINANT DNA TECHNOLOGY

SUBTOPIC : 8.3 Application of Recombinant DNA Technology
LEARNING OUTCOMES a) Briefly explain applications of Recombinant DNA Technology in mass
production of insulin using cDNA.
b) Describe the steps in production of insulin using cDNA.

MAIN IDEAS
EXPLANATION NOTES
/KEY POINT

Application Of • Many fields benefit from DNA technology and genetic engineering.
Recombinant - Agriculture
- Forensic
DNA
- Medicine – insulin for diabetics
Technology - Environment

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 cDNA is DNA molecule made in vitro using mRNA as a template

and the enzyme reverse transcriptase.
 A cDNA molecule lack introns compared to DNA of the genome
 Can be used directly to express the proteins.
Complementary  cDNA is often used to clone eukaryotic genes in prokaryotes
DNA (cDNA)
 cDNA is used because:
 cDNA represents the expression of the genes without the
introns.
 there is no process for RNA splicing in bacteria cell.
ntary DNA ( cDNA )
e
NA

fore
but
t in
e.

47

1. mRNA for insulin is isolated from the beta cells of islets of

Langerhans in the pancreas.

Production of 2. Reverse transcriptase enzyme is added to synthesis a cDNA by

insulin using mRNA as template. mRNA strand then discarded by using
mRNA-degrading enzyme.

3. DNA polymerase enzymes is added and synthesizes a second

DNA strand, complementary to cDNA in vitro.

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4. Restriction enzyme ( BamHI ) is used to cut the cDNA and isolate

just the sequence that encodes for the insulin protein.

5. Plasmid removed from a bacterial cell is cut at a specific site using

the same restriction enzyme (BamHI)

6. The cDNA is inserted to the plasmid using DNA ligase. The

recombinant plasmid carrying the human DNA for insulin.

7. The recombinant DNA then transform into the host(E. coli) by

transformation.

8. The bacterium E. coli with its recombinant plasmids, is allowed

to reproduce and undergo screening process.

9. Finally, a lot of bacterial clone carrying many copies of the gene

for insulin will be produced.

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