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BIOLOGY
Chapter14: pp. 249 - 263
10th Edition
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA probe array
tagged DNA did
bind to probe
DNA probe
Sylvia S. Mader
Biotechnology and
Genomics
tagged DNA did not
bind to probe
tagged
DNA
testing subject's DNA
PowerPoint® Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor
Copyright © The McGraw Hill Companies Inc. Permission required for reproduction or display
1
Outline

DNA Cloning

Recombinant DNA Technology
Restriction Enzyme
 DNA Ligase



Polymerase Chain Reaction
Biotechnology Products

Transgenic Bacteria, Plants, and Animals
Gene Therapy
 Genomics

2
DNA Cloning

Cloning is the production of identical copies of
DNA


Members of a bacterial colony on a petri dish are
clones because they all came from division of the
same cell.
Identical twins are clones


Single embryo separate to become two.
Gene cloning is production of many identical
copies of the same gene.



If the inserted gene is replicated and expressed, we
can recover the cloned gene or protein product.
Cloned genes have many research purposes
Humans can be treated with gene therapy
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Recombinant DNA Technology

Recombinant DNA (rDNA) contains DNA
from two or more different sources

Requires:

A vector



introduces rDNA into host cell
Plasmids (small accessory rings of DNA from bacteria) are
common vectors
Two enzymes are required to introduce foreign DNA
into vector DNA


A restriction enzyme - cleaves DNA, and
A DNA ligase enzyme - seals DNA into an opening
created by the restriction enzyme
4
Cloning a Human Gene
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human DNA
plasmid
bacterium
human cell
insulin gene
1. Restriction enzyme
cleaves DNA.
2. DNA ligase seals
human gene and
plasmid.
recombinant DNA
3. Host cell takes up
recombined plasmid.
4. Gene cloning occurs.
insulin
5
Restriction Enzyme




Cuts DNA at specific points.
Cleaves vector (plasmid) and foreign (human) DNA.
Cleaving DNA makes DNA fragments ending in short
single-stranded segments with “sticky ends.”
The “sticky ends” allow insertion of foreign DNA into
vector DNA.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA
duplex
A
G
A
A
T
T
C
G
C
T
C
T
T
A
A
G
C
G
T
T
restriction
enzyme
A
"sticky ends"
A
G
T
C
T
T
A
A
C
G
C
G
C
G
A
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DNA Ligase
Seals the foreign gene into the vector DNA
 Treated cells (bacteria) take up plasmids

Bacteria and plasmids reproduce.
 Many copies of the plasmid and many copies
of the foreign gene.

7
DNA Cloning: Polymerase Chain Reaction
(PCR)

Amplifies a targeted sequence of DNA


Create millions of copies of a single gene or a
specific piece of DNA in a test tube
Requires:

DNA polymerase


Withstands the temperature necessary to separate
double-stranded DNA.
A supply of nucleotides for the new,
complementary strand
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PCR
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PCR
cycles
DNA
copies
first
1
second
2
third
4
fourth
8
fifth
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DNA double strand
new strand
new
old strand
old
old
new
and so forth
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Applications of PCR: Analyzing DNA
Segments

DNA fingerprinting is the technique of
using DNA fragment lengths

Treat DNA segment with restriction enzymes
A unique collection of different fragments is
produced
 Gel electrophoresis separates the fragments
according to their charge/size
 Produces distinctive banding pattern

Usually used to measure number of
repeats of short sequences
 Used in paternity suits, rape cases, corpse
ID, etc.

10
DNA Fingerprinting & Paternity
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Mother
Child
Male 1
Male 2
Base repeat units
few
many
DNA Band patterns
Fluorescence units
a.
Increasing size
b. Automated DNA fingerprinting
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Biotechnology Products
Genetically engineered organisms can
produce biotechnology products.
 Organisms that have had a foreign gene
inserted into them are transgenic.

12
Transgenic Bacteria

Gene of interest is inserted into bacteria.
 Bacteria are grown in large vats called
bioreactors and product is harvested.

Products on the market include insulin, hepatitis B
vaccine, t-PA, and human growth hormone.

Transgenic bacteria can produce chemical
products.

Transgenic bacteria process minerals.

Metals Collection
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Transgenic Bacteria
Transgenic bacteria promote plant health


Bacteria that colonize corn roots can be
endowed with genes for insect toxin
Transgenic bacteria can degrade
substances.

Oil-Eating Bacteria
14
Genetically Engineered Bacteria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
(Both): Courtesy General Electric Research & Development
15
Transgenic Plants

Agricultural Crops
Foreign genes now give cotton, corn, and
potato strains the ability to produce an insect
toxin
 Soybeans are now resistant to a common
herbicide


Human Hormones

Plants are being engineered to produce human
proteins including hormones, clotting factors,
and antibodies in their seeds
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Transgenic Animals

Vortex Mixing:



Many types of animal eggs have taken up the gene for bovine
growth hormone (bGH)
The procedure has been used to produce larger fishes, cows,
pigs, rabbits, and sheep
Gene Pharming:




Use of transgenic farm animals to produce pharmaceuticals
Genes coding for therapeutic & diagnostic proteins are
incorporated into an animal’s DNA
The proteins appear in the animal’s milk
Plans are to produce drugs to treat



Cystic fibrosis
Cancer
Blood diseases, etc.
17
Transgenic Mammals
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
microinjection of human gene
human gene
for growth
hormone
donor of egg
development within
a host goat
human growth
hormone
Transgenic goat produces
human growth hormone.
milk
a.
transgenic goat cells
with gene for human
growth hormone
microinjection of
transgenic gene into
enucleated donor eggs
enucleated eggs
donor of eggs
development
within
host goats
milk
Cloned
transgenic
goats produce
human growth
hormone.
b.
18
Transgenic Animals

Researchers are using transgenic mice for
various research projects.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
one-celled
mouse embryos
with two X
chromosomes
no injection
inject SRY DNA
Embryo develops
into a female.
Embryo develops
into a male.
FEMALE
MALE
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Gene Therapy



Gene therapy involves procedures to give patients
healthy genes to make up for a faulty gene.
It also includes the use of genes to treat genetic
disorders and various human illnesses.
There are ex vivo (outside body) and in vivo (inside body)
methods of gene therapy.


Ex Vivo

Children with Severe Combined Immunodeficiency

Bone Marrow Stem Cells
In Vivo

Cystic Fibrosis

Nasal / Respiratory Spray
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Gene Therapy
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Brain
(gene transfer by injection)*
• Huntington disease
• Alzheimer disease
• Parkinson disease
• brain tumors
Skin
(gene transfer by modified
blood cells)**
• skin cancer
Lungs
(gene transfer by aerosol spray)*
• cystic fibrosis
• hereditary emphysema
Liver
(gene transfer by modified
implants)**
• familial hypercholesterolemia
Blood
(gene transfer by bone
marrow transplant)*
• sickle-cell disease
Endothelium
(blood vessel lining)
(gene transfer by
implantation of
modified implants)**
• hemophilia
• diabetes mellitus
Muscle
(gene transfer by injection)*
• Duchenne muscular dystrophy
Bone marrow
(gene transfer by implantation of
modified stem cells)**
• SCID
• sickle-cell disease
* in vivo
** ex vivo
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Genomics
Genomics is the study of genomes of
humans and other organisms.
 Sequencing the Bases

The Human Genome Project produced a
working draft of all the base pairs in all
chromosomes.
 Took 13 years to sequence three billion base
pairs along the length of chromosomes.

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Human Genome Project
Genome - All the genetic information of an
individual (or species)
 Goals of Human Genome Project

Determine the base pair sequence
 Construct a map showing sequence of genes
on specific chromosomes

23
Human Genome Project

Humans have 20,500 genes
Most code for proteins
 Much of the human genome was formerly
described as “junk”

Does not specify the order of amino acids in a
polypeptide
 Recent observation suggest that between 74% and
93% of the genome is transcribed into RNA


Thus, vast junk DNA wasteland may be much more
important than once thought
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Eukaryotic Gene Structure
Historically, genes were defined as discrete
units of heredity that corresponded to a
locus on a chromosome.
 Prokaryotes typically possess a single
circular chromosome.
 Eukaryotic chromosomes are much more
complex.

25
Chromosomal DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
intergenic sequences
Gene C
exon
exon
intron
DNA
Gene A
exon
Gene B
exon
pre-mRNA
intron
RNA
introns
5
3
Gene A mRNA
3
5
Gene B mRNA
5
3
Gene C mRNA
© Cindy Charles/PhotoEdit
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Eukaryotic Gene Structure

An Intergenic Sequences are DNA sequences
that occur between genes
 Repetitive DNA elements occur when the same
sequence of two or more nucleotides are
repeated many times along the length of one or
more chromosomes.
 Transposons are specific DNA sequences that
have the remarkable ability to move within and
between chromosomes.
27
Genomic Differences Between Species
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
28
HapMap Project

People inherit patterns of sequence differences,
called haplotypes



If one haplotype of a person has an A rather than a G
at a particular location in a chromosome, there are
probably other particular base differences near the A
Genetic data from African, Asian, and European
populations will be analyzed
A HapMap is a catalog common sequence
differences that occur in a species


The goal of the project is to link haplotypes to risk for
specific illnesses
May lead to new methods of preventing, diagnosing,
and treating disease
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Functional and Comparative Genomics


Functional genomics aims to understand the role of
genome in cells or organisms
DNA microarrays can monitor the expression of
thousands of genes simultaneously and tell us:




DNA microarrays contain microscopic amounts of known
DNA fixed onto a small glass slide
mRNA bind through complementary base pairing


What genes are turned on
Environmental conditions that turn on the gene
mRNA is produced by active cell.
Identify various mutations in the genome of an individual

This is called the person’s genetic profile.
30
DNA Microarray Technology
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA probe array
tagged DNA did
bind to probe
DNA probe
tagged DNA did not
bind to probe
tagged
DNA
testing subject's DNA
31
Genetic Profile
The complete genotype of an individual
 This is the person’s genetic profile
 A way of studying how genes work together
to control the phenotype

Analyze the genetic profile of many individuals
 Compare their profiles to their phenotypes

32
Proteomics

The study of the structure, function, and interaction of
cellular proteins
 At least 25,000 of our genes are translated into proteins
 The sum total of these proteins is called the human
proteome
 Understanding protein function is essential to the
development of better drugs



Correlate drug treatment to the particular genome
Increase efficiency and decrease side effects
Once the primary structure of these protein is known


It should be possible to predict their tertiary structure
Computer modeling of the tertiary of these proteins is an important
part of proteomics
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Bioinformatics


The application of computer technologies to the
study of the genome

Genomics and proteomics produce raw data

These fields depend on computer analysis to find
significant patterns in the data

Scientists hope to find relationships between genetic
profiles and genetic disorders
New computational tools will be needed to
accomplish these goals
34
Bioinformatics
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
intergenic sequences
Gene C
exon
exon
intron
DNA
Gene A
exon
Gene B
exon
pre-mRNA
intron
RNA
introns
5
3
Gene A mRNA
3
5
Gene B mRNA
5
3
Gene C mRNA
35
Review

DNA Cloning

Recombinant DNA Technology
Restriction Enzyme
 DNA Ligase


Polymerase Chain Reaction
Biotechnology Products
 Gene Therapy
 Genomics

36
BIOLOGY
Chapter14: pp. 249 - 263
10th Edition
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA probe array
tagged DNA did
bind to probe
DNA probe
Sylvia S. Mader
Biotechnology and
Genomics
tagged DNA did not
bind to probe
tagged
DNA
testing subject's DNA
PowerPoint® Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor
Copyright © The McGraw Hill Companies Inc. Permission required for reproduction or display
37