BIO 3202

RECOMBINANT DNA TECHNOLOGY

Programme Core Course

4 (3+1)
School of Fundamental Science
Assoc. Prof Dr. Cha Thye San
Dr. Malinna Jusoh

Course Synopsis
This course discusses the principles of recombinant
DNA technology, which covers
gene cloning, molecular enzymes,
electrophoresis, PCR techniques,
cDNA
and genomic library construction, gene
expression study, DNA sequence analysis, DNA
binding protein, bioinformatic and genetic
transformation technology. The students will have
intensive hand-on practicals on basic recombinan
DNA techniques

Assessment
Continuous assessment 60 %

Laboratory report (20%)

Assignments 1 (10 %)
Assignments 2 (10 %)
Test (20 %)
Final exam 40 %

Program
Learning
Outcome (PLO)

Course Learning Outcome (CLO)

Learning
Domain
C P A

Teaching & Learning Activities

S
C
L

C
a
s
e

M
o
d
u
l
e

S
t
u
d
y
Menerangkan prinsipprinsip dalam teknologi
DNA rekombinan dengan
tepat
Melaksanakan
PLO2
Technical Skills/ eksperimen teknologi
Psychomotor
DNA rekombinan dengan
menggunakan peralatan
saintifik yang sesuai.
Menghubungkaitkan
PLO3
CTPS
teknik-teknik yang
digunakan dalam
teknologi DNA
rekombinan dengan
berkesan.
Menggunakan sumber
PLO7
Lifelong Learning bioinformatik yang sesuai
& Info Mgmt
untuk membincangkan
contoh-contoh dan
aplikasi dalam teknologi
DNA rekombinan.
PLO1
Knowledge

T
u
t
o
r
i
a
l

L
a
b

D
is
c
u
s
si
o
n

G
r
o
u
p
w
o
r
k

L
e
c
t
u
r
e

L
i
t
S
e
a
r
c
h
/

Assessment Tasks &

Weightage
T P
P
R
Fi
e r
ro
e
n
s o
je
p
al
t
j
ct
or
E
1 e
(2
t
x
c
)
a
t
m
(
1
)
20
20

10/
10

10

20

3/7

Course Outline

Week
1

Lecture Title
Introduction to Recombinant DNA (rDNA)
Technology

Hour
3

Milestones in DNA history

The basics of rDNA
Advantages and disadvantages of rDNA
technology
Examples of rDNA technology

Week
2

Lecture Title
Enzyme for DNA Manipulation in rDNA
Restriction enzymes
Ligation enzymes
DNA synthesis enzymes
Other modification enzymes

Hour
6

Week
4

Lecture Title
Gene Cloning

Hour
9

Extraction and purification of nucleic acids

Ligation
Transformation
Plasmid vectors
Selection of recombinant plasmid

Week
7

Lecture Title
Polymerase Chain Reaction (PCR)
Principles of PCR
PCR technique
Primer design
Reverse-transcription PCR and quantitative
real-time PCR
Applications of PCR

Hour
6

Week
9

Lecture Title
Genomic and cDNA Libraries

Hour
3

Genomic libraries
cDNA libraries
Construction and evaluation of library
Applications of library

Week
10

Lecture Title
Analysis of Gene Expression
Northern blots
Real-time PCR
RNA-seq

Hour
3

Week
11

Lecture Title
DNA Sequencing

Hour
3

Principles of DNA sequencing

Analysis of 3 and 5 UTR
Identification of start/stop codons, exons,
introns and transcription factors

Week
12

Lecture Title
Bioinformatics
Gene and protein databases
Genome browsers
Comparing genomes of rice, Arabidopsis,
bacteria etc
Web-based analysis

Hour
6

Week
14

Lecture Title
Latest application in rDNA technology
Examples of rDNA technology
Latest advance in rDNA technology

Hour
3

References
Dale, J.W., Von Schantz, M. & Plant, N. 2012. From Genes To
Genomes: Concepts and Applications of DNA Technology. John
Wiley & Sons Ltd, UK.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G.,
Smith, J.A., Struhl, K. 2002. Short Protocols In Molecular Biology :
A Compendium of Methods From Current Protocols In
Molecular Biology, (5th edition). VOL 1 & 2. New York, Wiley & Son Inc.
Brown, T.A. 2002. Genome (2nd edition). John Wiley & Sons, INC.
Publication, New York.
Klug, W.S., Cummings, M.R. & Spencedr, C.A. 2007. Essentials of
Genetics (6th ed). Pearson Prentice Hall. New Jersey.
Sambrook, J and Russell, D.W. 2001. Molecular Cloning: A
Laboratory Manual, (3rd edition). VOL 1, 2 & 3. Cold Spring Harbor,
N.Y.: Cold Spring Harbor Laboratory Press

Lecture 1
Introduction to Recombinant
DNA Technology

The Basics of Recombinant DNA

Recombinant DNA (rDNA)
DNA that has been created artificially.
From two or more sourcesincorporated into a
single recombinant molecule
sequences that would not normally occur together
differs from genetic recombinationdoes not
occur through natural processes within the cell,
but is engineered

 DNA
codes genetic information for the transmission of
inherited traits
made up of a base consisting of sugar, phosphate and
one nitrogen base
four nitrogen bases, adenine (A), thymine (T), guanine
(G), and cytosine (C)
nitrogen bases are found in pairs, with A & T and G & C
sequence of the nitrogen bases=double helix structure
sequence and number of bases =creates diversity

Remember!
As a biological
sciences student,
you should be
able to draw the
structure of DNA
like this

Milestones in DNA History

1869: Johann Friedrich Miescher identifies a weakly acidic substance of

unknown function in the nuclei of human white blood cells. This
substance will later be called deoxyribonucleic acid, or DNA.
1912: Physicist Sir William Henry Bragg, and his son, Sir William
Lawrence Bragg, discover that they can deduce the atomic structure
of crystals from their X-ray diffraction patterns. This
scientific
tool was the key in helping Watson and Crick determine DNA's
structure.
1924: Microscope studies using stains for DNA and protein show that
both substances are present in chromosomes.
1928: Franklin Griffith, a British medical
officer, discovers that genetic
information can
be transferred from heat-killed bacteria cells to
live ones. This phenomenon, called
transformation,
provides the first evidence
that the
genetic material is a heat-stable
chemical.

1944: Oswald Avery, and his colleagues Maclyn McCarty and Colin
MacLeod, identify Griffith's transforming agent as DNA. However, their
discovery is greeted with skepticism, in part because many scientists still
believe that DNA is too simple a molecule to be the genetic material.
1949: Erwin Chargaff, a biochemist, reports that DNA composition is
species specific; that is, that the amount of DNA and its nitrogenous
bases varies from one species to another. In addition, Chargaff finds that
the amount of adenine equals the
amount of thymine, and the amount
of guanine equals the amount of cytosine in DNA from every species.

1953: James Watson and Francis Crick

discover the molecular structure of DNA.
1962: Francis Crick, James Watson, and
Maurice Wilkins receive the Nobel Prize for
determining the molecular structure of DNA.

Milestones in Biotechnology

1909: British physician Archibald Garrod first proposes the relationship

between genes and proteins. He hypothesizes that genes might be
involved in creating the proteins that carry out the chemical reactions of
metabolism.
1930s: Through experimentation with mutant strains of Neurospora
bread mold, George Beadle and Edward Tatum support Garrod's
hypothesis. This evidence will give rise to the one gene-one protein
hypothesis, that each protein in a cell results from the expression of a
single gene.

1957: During a dysentery epidemic in

Japan, biologists discover that
some strains of bacterium are
resistant to antibiotics. Later
scientists will find that this resistance
is transferred by
plasmids.

1961: Sidney Brenner and Francis Crick establish that groups of three
nucleotide bases, or codons, are used to specify individual amino
acids.
1966: The genetic code is deciphered when biochemical analysis
reveals which codons determine which amino acids.
1970: Hamilton Smith, at Johns Hopkins Medical School, isolates the
first restriction enzyme, an enzyme that cuts DNA at a very specific
nucleotide sequence. Over the next few years, several
more
restriction enzymes will be isolated.
1972: Stanley Cohen and Herbert Boyer
combine their efforts to create
recombinant DNA. This technology will be
the beginning of
the biotechnology
industry.
1976: Herbert Boyer cofounds
Genentech, the first firm founded in the
United States to apply recombinant DNA
technology

1978: Somatostatin, which regulates human growth hormones, is the

first human protein made using recombinant technology.

Biographies

Francis H. C. Crick

He received his college degree in

physics and was starting graduate
school when the World War II began.

During the war, Crick worked on

weapons for the British Admiralty.

He was in his late 20s by the time the

war ended, but he decided to go back
to school for a PhD.

He went to the Cavendish Laboratory of Cambridge University to

pursue this interest by studying proteins.

In 1951, James Watson arrived at Cavendish, and the two began the
collaboration that would lead to the discovery of the structure of the
DNA molecule.

Before Crick received his PhD, he completed the work that would earn
him a Nobel Prize.

James D. Watson

As a boy, James Watson was already

very interested in science, particularly
in birds.

First picked up as a senior in college,

to learn about the gene.

Got into graduate school at Indiana

University, he decided to study the
simplest form of life bacteria to
understand genes.

To Europe, as a postdoctoral fellow, to learn more about

biochemistry and bacteriophages.

In 1953, Watson and Crick sparked a revolution with their discovery

of the helical structure of the DNA molecule.

Watson was only 25 years old when their findings were published.

He was only 34 when he was awarded the Nobel Prize.

Herbert W. Boyer

12 years old, he thought he wanted to be a

professional football player.

Science teacher, helped change Boyer's

mind.

Went to St. Vincent's College to study

biology and chemistry.

Received both his MS and PhD degrees in

bacteriology.

By 1966, Boyer had found his way to California, where he began work
as an assistant professor at the University of California San Francisco.

1972, Boyer met Stanley Cohen, and together they pioneered the field
of recombinant DNA.

Their work led to the founding of biotechnology firms such as

Genentech.

Stanley N. Cohen

Grew up in Perth Amboy, New Jersey, a

little town about 30 miles from New York
City.

As a boy, he was interested in atomic

physics, but a biology teacher in high
school inspired his interest in genetics.

He went on to study biology and then

medicine.

In 1968, Cohen went to Stanford University to work as both a

researcher and a physician. Began to explore the field of bacterial
plasmids.

Wanted to understand how the genes on plasmids could make

bacteria resistant to antibiotics.

1972, Cohen's investigations, combined with those of Herbert Boyer,

led to the development of methods to combine and transplant genes.

This discovery signaled the birth of genetic engineering.

How is Recombinant DNA made?

There are three different methods by which
recombinant DNA is made:
Transformation
Phage introduction
Non-bacterial transformation

Transformation

Phage introduction

Non-bacterial transformation:
microinjection

How does
rDNA work?

Why is rDNA important?

Improved medicines
Improved livestock (resistance to disease)
Improved crops (resistance to disease, higher
yields)
Prevention of genetic diseases
Lowering the cost of medicines (i.e. Insulin)
Safer medicines (i.e. Insulin)
Treatment for pre-existing conditions (i.e.
Cancer)

The disadvantages of rDNA Technology

Safety concerns (viruses developing antibiotic
resistance)
Environmental concerns (developing resistance
to fungi)
Ethical dilemmas over human treatment
Potential for experimental abuse (doctors using
patients as test subjects)
Germline treatment going from treating diseases
to a method for choosing the traits you want in a
child

Genetic Engineering
+
Recombinant DNA Technology
=
Endless Possibilities

Examples of rDNA Technology

1. Insulin

For many years, insulin extracted from the

glands of cows and pigs was used
Human insulin produced by bacteria or yeast
(biosynthetic insulin) that is genetically
compatible with diabetic patient

2. Vaccines (Hepatitis B)
composed of viral protein manufactured by
yeast cells, which have been recombined
with viral genes
the vaccine is safe because it contains no
viral particles

3. Gene therapy
a recombinant DNA process in which cells
are taken from the patient, altered by
adding genes, and replaced in the patient,
where the genes provide the genetic codes
for proteins the patient is lacking
E.g. melanoma and adenosine deaminase
(ADA)

4. Agricultural

Golden riceis a variety of Oryza sativa rice

produced through genetic engineering to
biosynthesize beta-carotene, a precursor of
vitamin A
Genetically modified cropsherbicide-resistant
crops, insect-resistant crops

pronounced "flavor saver

polygalacturonase (PG)

End of Lecture 1
Thank You