Gene Biotechnology

Through gene manipulation (ultimately leads to change in protein

expression), one may:
1) Introduce foreign gene(s) into cells / organisms, aiming:
A) to change the characteristic of an organism, eg,
- confer disease resistance to plants,
- increase growth rate, increase nutritional contents,
- to modify a micro-organism that can remove toxins
from the environment.
B) to massively produce the corresponding gene product(s)
(proteins) for use, eg,
- production of insulin in yeast,
- production of anti-thrombin (protein that prevents blood
clots) from cow milk
2) Replacing a defective gene in the organism (human)
- gene therapy of adenosine deaminase (ADA) deficiency
(genetic disease characterized by immunodeficiency).

In this lecture:

Gene Biotechnology
We will learn the ways to manipulate DNA:
- Cloning
- Sequencing
- Polymerase Chain reaction
Then we will learn some applications of these gene biotechniques.
- Human genome project
- DNA profiling
- Personalized medicine

Gene Technology Genetic engineering or Genetic modification

To manipulate gene for biotechnology applications, it is

required to:
1) Read the genes and understand their functions.
2) Supplement the useful / remove the defective genes in
the organisms

How to read the genome ?

In early days (20-30 years ago)
- The main task was to read the genome.
- However, the genome is too huge for
direct analysis ( ~100 to 200 million base
pairs (letters) in each chromosomes), 6
billion base pair in total.
- Moreover, each gene only has 2 copies
in a cell.
- Smallest bacteria genome: 139 kbp.
Solution?
- By DNA cloning and sequencing

 DNA cloning

is a set of experimental methods in molecular biology that are used to
assemble recombinant DNA molecules and to direct their replica9on within
host organisms.
Cloning: refers to the fact that the method involves the replica9on of a
specic DNA fragment star9ng from a single living cell to generate a large
popula9on of cells containing iden9cal DNA molecules.
Molecular cloning generally uses DNA sequences from two dierent
organisms: the species that is the source of the DNA to be cloned, and the
species that will serve as the living host for replica9on of the recombinant
DNA.
Molecular cloning methods are central to many contemporary areas of
modern biology and medicine

DNA Cloning

Two important tools:

Restric9on enzymes
Plasmids

Restric0on enzymes: molecular scissors that can cut double-stranded or

single stranded DNA at specic recogni9on nucleo9de sequences known as
restric9on sites.
- Found in bacteria for the defense against invading viruses.

<hMp://nobelprize.org/nobel_prizes/
medicine/laureates/1978/>

Restric0on enzymes

cut DNA at specic nucleo9de sequences (restric9on sites), and
producing pieces of DNA called restric9on fragments with s9cky ends
important for joining DNA from dierent sources.
used together with DNA ligase, an enzyme that connects the DNA pieces
into con9nuous strands by forming bonds between adjacent nucleo9des.
DNA pieces can be cut and joined according to design (similar to cut and
paste in MicrosoW Word)

 2013 Pearson Education, Inc.

Recombinant DNA technology

Recognition site (recognition sequence)
for a restriction enzyme
DNA
1 A restriction enzyme cuts the

Restriction
enzyme

DNA into fragments.

ky
Stic

(Scissor)

end
Stic

ky e

nd

A DNA fragment is added from

another source.

3 Fragments stick together by

base pairing.

DNA ligase joins the fragments

into strands.

DNA
ligase

(Glue)
Recombinant DNA molecule

Molecular (DNA) cloning and recombinant DNA technology

Plasmid: A plasmid is a DNA molecule that is separate from, and can replicate
independently of, the chromosomal DNA. They are double stranded and, in
many cases, circular. Plasmids usually occur naturally in bacteria, but are
some9mes found in eukaryo9c organisms.
Discovered by Japanese scien9st Wanatabe in 1960 for its role in an9bio9c
resistance in a strain of bacterium Shigella that defeated three kinds of
an9bio9cs.

Bacterial
chromosome
Remnant of
bacterium

Colorized TEM

Plasmids

 Plasmids are suitable for recombinant DNA technology

because they
can carry virtually any gene,
are small in size, easy to manipulate
can act as vectors, DNA carriers that move genes from one cell to
another, and
are ideal for gene cloning, the produc9on of mul9ple iden9cal copies
of a gene-carrying piece of DNA.

2013 Pearson Education, Inc.

An example of a very popular plasmid in 1970s, pBR322

What makes a good vector?

Size small enough for easy
sepera9on from genomic DNA
Origin of replica9on the site
for plasmid DNA to replicate
separately from host cells
chromosome.
Mul9ple Cloning sites
Selec9on marker genes
T7 promoter sequence for
expression vectors
<R. Renneberg; Biotechnology for Beginners, 2008 >

Applications of DNA cloning:

- Amplification of DNA.
- When a gene is fused to a
suitable promoter (switch), the
gene can be expressed in a
given organism to massively
produce the desired protein.

An example of plasmid

http://www.youtube.com/watch?v=juP6iHMIYkE

DNA sequencing: A technique to read the genome

Materials required for DNA sequencing reactions:

- deoxyribonucleotides (dATP, dCTP, dGTP, dTTP)
- dideoxyribonucleotide (fluorescence dye labeled)(ddATP,
ddCTP, ddGTP, ddTTP)
- DNA polymerase (an enzyme that joins nucleotides together).
- A primer (to start the synthesis)

<Albert Bruce, Molecular Biology of the Cell>

http://www.youtube.com/watch?v=lgASqWbemCc
http://www.youtube.com/watch?NR=1&v=6ldtdWjDwes
http://www.youtube.com/watch?v=91294ZAG2hg

<Albert Bruce, Molecular Biology of the Cell>

Recombinant DNA & Gene Biotechnology

Signicance of PCR-based DNA-sequencing technology
Dye Terminator:fluorescent dye on ddNTP

Industrial automation

Genomes sequenced
E. Coli
C. Elegans
Rice

Applied Biosystems
96-capillary 3730xl DNA sequencer

Horse
Human

DNA Sequencing (Nobel Prize in Chemistry 1980)

PCR-based Recombinant DNA technology

The PCR method a copying machine for DNA molecules

is a technique to copy quickly and precisely a specic segment of DNA
(up to a million copies per hour) and
can generate enough DNA, from even minute amounts of blood or
other 9ssue, for cloning and sequencing, etc.

PCR-based Recombinant DNA technology

The pressing ques0on that prompted PCR technology: how to get more DNA?

DNA always exists in

small amount in a cell,
how to get more DNA
with the same gene9c
informa9on?

The answer: Learn from
nature, learn from DNA
replica9on

PCR-based Recombinant DNA technology

The PCR method a copying machine for DNA molecules

http://www.youtube.com/watch?v=2KoLnIwoZKU

PCR-based Recombinant DNA technology: a quick review

Polymerase Chain Reac9on (1993 Nobel Prize)

4 key components: template, primer, DNA polymerase, dNTPs

Recombinant DNA & Gene Biotechnology

PCR ow chart

<Albert Bruce, Molecular Biology of the Cell>

Recombinant DNA & Gene Biotechnology

Signicance of PCR: amplica0on of DNA
DNA molecules can be mass-produced from incredibly small amounts of
material with PCR. This has enabled us to characterize and compare the
gene9c material from dierent individuals and organisms.
1993 Sci-Fi movie
Directed by Steven Spielberg
won 3 Oscars, and life9me
worldwide box oce revenue of
$914,691,118

Dinosaur blood in mosquito found
trapped in ancient fossil amber

PCR
Dinosaur DNA


Ostrich eggs
Dinosaur

PCR-based site-directed mutagenesis

- A way to change the nucleo0de sequence of a gene

Site-directed mutagenesis

A way to change the gene sequence

<Albert Bruce, Molecular Biology of the Cell>

Site-directed mutagenesis ow chart

HK Polytechnic University

Y9srevinu

HK Polytechnic Y9srevinu

Signicance of Site-directed Mutagenesis

This technology has enabled scien9sts to understand the
func9onal importance of individual nucleo9de (DNA or
RNA) or amino acid residue (protein) by altering them one
at a 9me.

This technology makes molecular engineering a reality.
Tailor-made DNA, RNA and protein molecules with their
natural bases or amino acids modied for func9onal design
can be produced.

The Human Genome Project:

In 1984
the U.S. Department of Energy (DOE), National
Institutes of Health (NIH), and international groups
held meetings about studying the human genome.
In 1988
The US National Research Council recommended
starting a program to map the human genome.
In 1990
NIH and DOE published a plan for the first five years
of an expected 15-year project.
The project would develop technology for analyzing
DNA; map and sequence human and other genomes
including fruit flies and mice; and study related ethical,
legal, and social issues.
In 2001
the Human Genome Project international consortium published a first draft and initial
analysis of the human genome sequence. A wealth of information was obtained from the
initial analysis of the human genome draft.
For instance, the number of human genes was estimated to be about 30,000 (later revised
to about 20,000). Researchers also reported that the DNA sequences of any two human
individuals are 99.9 percent identical.

Human genome project

International scientific research project.
Goal: determining the sequence of chemical base pairs which
make up human DNA, and of identifying and mapping all of the
genes of the human genome.
The world's largest collaborative biological project:
Proposed and funded by the US government; planning started in
1984, the project got underway in 1990, and was declared complete
in 2003.

Major findings and applications for Human Genome projects

http://www.genome.gov/Pages/Education/AllAbouttheHumanGenomeProject/
GuidetoYourGenome07_vs2.pdf

Application of genome sequencing

1) Genetic tests
- Obtain DNA from blood.
- Test if it contains specific mutation.
- Predicts / Determine if offspring will
inherit disease gene from parents.

Future:
- Can predict the risk of getting certain
cancers, diabetes, heart disease, etc.
- Prevention of diseases.

2) Drug development using genomic data

- Genomic data leads to the development of better drugs.
- Drug development in the past: random screening of
chemicals against a disease.
- Now can sue genomic information to design drugs
targeted at specific pathways involved in the disease.
- Hope: new drugs will work better and have fewer side
effects

3. Prediction of drug response

4) Understand the gene make up of other organisms

It took almost 20 years to sequence human genome.

But within these several years many of the known organisms has been sequenced.

Why?

Because sequencing technology has been much improved

Old DNA sequencing

machine
New technology: Massively parallel sequencing

New technology monitors hundred thousands reaction at the same time

It took ~20 years to sequence the first human genome. Now it takes only a week

5) In Forensic and Parentage analysis

 FBIs Combined DNA Index System (CODIS)

Have chosen 13 unique STRs in human genome for
iden9ca9on and
Have built up a database of convicts, suspects etc.

< www.cstl.nist.gov/strbase/images/codis.jpg >

Crime scene DNA

STR site 2

STR site 1
AGAT

GATA

Different numbers of
short tandem repeats

Same number of
short tandem repeats

AGAT
Suspects DNA

GATA

Amplified
crime scene
DNA

Amplified
suspects
DNA

Longer
fragments

Shorter
fragments

Summary
Recombinant DNA is constructed when scien9sts
combine pieces of DNA from two dierent sources
to form a single DNA molecule.
Recombinant DNA is produced by combining two
ingredients:
1. a bacterial plasmid and
2. the gene of interest.

Molecular cloning is the process to generate

recombinant DNA.

DNA technology: applica0ons

<W.J. Thieman and M.A. Palladino; Introduc9on to Biotechnology, 2009 >