Lecture 2- Gene Biotechnology

7/24/2019 Lecture 2- Gene Biotechnology

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Gene Biotechnology


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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).


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Gene Biotechnology

In this lecture:

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


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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 inthe organisms

Gene Technology Genetic engineering or Genetic modification


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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), 6billion 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

How to read the genome ?


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DNA Cloning


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Recognition site (recognition sequence)

for a restriction enzyme

Restrictionenzyme

Stickye

nd

Stickyend

DNA

DNAligase

Recombinant DNA molecule

A DNA fragment is added fromanother source.

A restriction enzyme cuts theDNA into fragments.

Fragments stick together by

base pairing.

DNA ligase joins the fragments

into strands.

1

2

3

4

Recombinant DNA technology

(Scissor)

(Glue)


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Bacterial

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Applications of DNA cloning:

-

Amplification of DNA.

- When a gene is fused to asuitable promoter (switch), the

gene can be expressed in a

given organism to massivelyproduce the desired protein.

An example of plasmid


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


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DNA sequencing: A technique to read the genome


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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)

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


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Dye Terminator:fluorescent dye on ddNTP

Industrial automation

Applied Biosystems

96-capillary 3730xl DNA sequencer

Genomes sequenced

E. Coli

C. Elegans

Rice

Horse

Human

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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 yearsof 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 2001the 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.


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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.


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

Major findings and applications for Human Genome projects


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1) Genetic tests

- Obtain DNA from blood.

- Test if it contains specific mutation.

- Predicts / Determine if offspring willinherit disease gene from parents.

Future:

- Can predict the risk of getting certaincancers, diabetes, heart disease, etc.

- Prevention of diseases.

Application of genome sequencing


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2) Drug development using genomic data

- Genomic data leads to the development of better drugs.

-

Drug development in the past: random screening ofchemicals against a disease.

-

Now can sue genomic information to design drugstargeted at specific pathways involved in the disease.

-

Hope: new drugs will work better and have fewer side

effects


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3. Prediction of drug response


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It took almost 20 years to sequence human genome.But within these several years many of the known organisms has been sequenced.

Why?

4) Understand the gene make up of other organisms


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Because sequencing technology has been much improved

Old DNA sequencingmachine

New technology: Massively parallel sequencing

New technology monitors hundred thousands reaction at the same time


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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


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5) In Forensic and Parentage analysis


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Crime scene DNA

Suspect

s DNA

Same number ofshort tandem repeats

Different numbers ofshort tandem repeats

STR site 1 STR site 2

AGAT

AGAT GATA

GATA


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Amplified

crime sceneDNA

Amplified

suspectsDNA

Longerfragments

Shorterfragments


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Summary

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Lecture 2- Gene Biotechnology

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