BIOL0011

Lecture 24

GENETIC ENGINEERING
Introduction & Restriction enzymes

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OBJECTIVES
You must be able to:
Define: Restriction Enzymes (RE’s); Palindromes; Restriction Mapping
Outline how RE’s work

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Introduction to Enzymology History
• 1955 Arthur Kornberg and colleagues isolated DNA polymerase
• 1966 B. Weiss and C.C. Richardson isolated DNA ligase
• 1968 H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterized
the first sequence specific restriction enzyme
• Phage growth restriction
• Phages grow and infect one strain, but not the other strain
• Methylation: Not modified get degraded by hosts enzymes
• Methylase and Nuclease

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 Introduction to Enzymology History
• Late 1960s, Stewart Linn and Werner Arber isolated two types of enzymes
responsible for phage growth restriction in E. coli bacteria.
• Methylase cleaved methylated DNA
• Restriction nuclease cleaved unmethylated DNA at a wide variety of sites
• Site-specific nuclease
• 1968, Johns Hopkins University, H.O. Smith, K.W. Wilcox, and T.J. Kelley first
restriction nuclease whose functioning depended on a specific DNA
nucleotide sequence
• HindII from Haemophilus influenzae bacteria
• Always cut at a particular point within a specific sequence of six base pairs
• 5 GTAC 3
• 3 CATG 5

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Genetic Engineering: Recombinant DNA
Technology
Recombinant DNA technology :

The manipulation and combination of DNA molecules from different

sources

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 Genetic Engineering: Techniques Used
1. Cleavage of DNA at specific sites by restriction enzymes greatly facilitates the
isolation and manipulation of individual genes.
2. Gel electrophoresis: Separation and analyses of macromolecules based on their
size and charge
3. DNA cloning either through the use of cloning vectors and the polymerase
chain reaction, whereby a single DNA molecule can be copied to generate many
billions of identical molecules.
4. Nucleic acid hybridization makes it possible to find a specific sequence of DNA or
RNA with great accuracy and sensitivity on the basis of its ability to bind a
complementary nucleic acid sequence.
5. Sequencing of all the nucleotides in a purified DNA fragment makes it possible to
identify genes and to deduce the amino acid sequence of the proteins they encode.
6. Simultaneous monitoring of the expression level of each gene in a cell, using
nucleic acid microarrays that allow tens of thousands of hybridization reactions to be
performed simultaneously.
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Summary: 3 Major Steps
• Restriction Enzyme use: Recombinant DNA technology requires the
use of molecular scissors called restriction enzymes, which cut DNA at
specific sequences.

• Gel Electrophoresis: Separation and analyses of macromolecules

• DNA Cloning: Two methods –

• Use of plasmids
• PCR

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What are Restriction Enzymes?
• Restriction enzymes are DNA-cutting enzymes found
in bacteria as a defense against invasion from foreign DNA (and
harvested from them for use).
• Also called: Restriction endonucleases
• A restriction enzyme recognizes and cuts DNA only
at a particular sequence of nucleotides.
• Bacteria prevent their own DNA from being cut by
modifying their nucleotides via DNA methylation.

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How Restriction Enzymes work
• RE cleave DNA at any site where a SPECIFIC short sequence of
nucleotide occurs

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Palindromes
While recognition sequences vary widely, with lengths between
4 and 8 nucleotides, many of them are palindromic. That is,
the sequence on one strand reads the same in the reverse
direction on the complementary strand.

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How do Restriction Enzymes work?
• Blunt ends: Cleave opposing phosphodiester bonds no unpaired
bases on ends

• Sticky ends: Staggered cuts of two DNA strands leaving unpaired

nucleotides at the ends

• Base-pair with complementary sticky ends

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Types of Restriction Enzymes
• Type I cleave DNA at random sites that can be more than 1,000 bp
from the recognition sequence.
• Type II first isolated in 1970 (Hamilton Smith), require no ATP and
cleave DNA within the recognition sequence.
• Type III cleave about 25 bp from the recognition sequence.

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Nomenclature
• This follows a general pattern:
• 1. The first letter of the name of the genus in which a given enzyme is discovered, is
written in capital letters.

• 2. This is followed by the first two letters of the species name of the organism. All three
letters are written in italics.
• Eg Eco from Escherichia coli; Hin from Haemophilus influenza

• 3. The strain or identification type is depicted as a subscript

• 4. All RE are designated by the general symbol R, prefixed to their name

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Named Example
EcoRI

E genus Escherichia
co species coli
R strain RY13
I first endonuclease identified

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Restriction Mapping
• This is the process of obtaining structural information on a piece of
DNA by the use of restriction enzymes.
• A restriction map is a description of restriction endonuclease cleavage
sites within a piece of DNA.
• Restriction mapping involves digesting DNA with a series of restriction
enzymes and then separating the DNA fragments by agarose gel
electrophoresis.

• The distance between enzyme sites can be determined by the size of

the DNA fragments.
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Restriction Maps
• Show the specific RE recognition sites

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Restriction Mapping
• Multiple RE’s used
• Each RE cuts at a different location
• Fragments are separated by gel electrophoresis and move through
the gel according to size
• Each RE fragment creates a signature band

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Uses of Restriction Enzymes
• Creating Genome Libraries – Recombinant DNA Technology

• Cloning DNA molecules

• Studying nucleotide sequences

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 GENETIC ENGINEERING
Recombinant DNA Technology

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What is recombinant DNA technology?
• Recombinant DNA technology: joining together of
DNA molecules from two different species that are
inserted into a host organism to produce new genetic
combinations that are of value to science, medicine
industry and agriculture.

• (Encyclopedia Britannica)

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REMINDER…….
Central to the technology are the following key techniques:
1. Cleavage of DNA at specific sites by restriction enzymes greatly facilitates the
isolation and manipulation of individual genes. (Covered previously)
2. DNA cloning either through the use of cloning vectors and the polymerase
chain reaction, whereby a single DNA molecule can be copied to generate many
billions of identical molecules.
3. Nucleic acid hybridization makes it possible to find a specific sequence of DNA or
RNA with great accuracy and sensitivity on the basis of its ability to bind a
complementary nucleic acid sequence.
4. Sequencing of all the nucleotides in a purified DNA fragment makes it possible to
identify genes and to deduce the amino acid sequence of the proteins they encode.
5. Simultaneous monitoring of the expression level of each gene in a cell, using
nucleic acid microarrays that allow tens of thousands of hybridization reactions to be
performed simultaneously.

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Gel Electrophoresis: Isolation of DNA
fragments
Gel electrophoresis is a method for separation and analysis of
macromolecules (DNA, RNA and proteins) and their fragments, based
on their size and charge.
It is used in molecular biology to separate a mixed population of DNA
and RNA fragments by length, to estimate the size of DNA and RNA
fragments or to separate proteins by charge.

(Modified from Wikipedia)

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Gel electrophoresis apparatus
• Gel electrophoresis apparatus – an agarose gel is placed in this buffer-filled box
and an electrical field is applied via the power supply to the rear. The negative
terminal is at the far end (black wire), so DNA migrates toward the cathode (red
wire).

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Electrophoresis: Results

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Electrophoresis: Initial interpretation of
results

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DNA Cloning: Generates billions of identical molecules from
a single DNA molecule produced using RE’s and electrophoresis

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DNA Cloning: Polymerase Chain Reaction (PCR)
• PCR is based on using the ability of DNA polymerase to synthesize
new strand of DNA complementary to the offered template strand.
• Because DNA polymerase can add a nucleotide only onto a
preexisting 3'-OH group, it needs a primer to which it can add the first
nucleotide.
• This requirement makes it possible to delineate a specific region of
template sequence that the researcher wants to amplify.
• At the end of the PCR reaction, the specific sequence will be
accumulated in billions of copies (amplicons).

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PCR: Requirements
• DNA Template

• DNA Polymerase

• Primers

• Nucleotides

• Reverse Transcription- PCR

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How PCR Works:

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Summary: 3 Major Steps
• Restriction Enzyme use: Recombinant DNA technology requires the
use of molecular scissors called restriction enzymes, which cut DNA at
specific sequences.

• Gel Electrophoresis: Separation and analyses of macromolecules

• DNA Cloning: Two methods –

• Use of plasmids
• PCR

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Genetic Engineering: Applications
• Medicine: Production of pharmaceuticals & Gene Therapy

• Agriculture: Plants and Animal Husbandry

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Uses of Recombinant DNA Technology
• Recombinant DNA technology has applications in health and
nutrition.

• In medicine, it is used to create pharmaceutical products such as

human insulin and in gene therapy.

• In agriculture, it is used to impart favorable characteristics to plant to

increase their yield and improve nutritional content and in animal
husbandry.

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Creating Pharmaceutical Products: eg Human
Insulin

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Other Pharmaceutical Products
• Tumor necrosis factor. Treatment for certain tumor cells
• Interleukin-2 (IL-2). Cancer treatment, immune deficiency, and HIV
infection treatment
• Prourokinase. Treatment for heart attacks
• Taxol. Treatment for ovarian cancer
• Interferon. Treatment for cancer and viral infections
• In addition, a number of vaccines are now commercially prepared
from recombinant hosts.

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Gene Therapy
• Gene therapy is an experimental technique that uses genes to treat or
prevent disease. In the future, this technique may allow doctors to
treat a disorder by inserting a gene into a patient’s cells instead of
using drugs or surgery. Researchers are testing several approaches to
gene therapy, including:
• Replacing a mutated gene that causes disease with a healthy copy of
the gene.
• Inactivating, or “knocking out,” a mutated gene that is functioning
improperly.
• Introducing a new gene into the body to help fight a disease.
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Gene Therapy
• Although gene therapy is a promising treatment option for a number
of diseases (including inherited disorders, some types of cancer, and
certain viral infections), the technique remains risky and is still under
study to make sure that it will be safe and effective. Gene therapy is
currently only being tested for the treatment of diseases that have no
other cures.

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Plant Genetic Engineering
• A: Crop Improvement
• B: Genetically engineered traits: Herbicide, insect & virus resistance;
altered oil content; delayed fruit ripening; pollen control
• C: Weather resistant crops
• D: Genetically engineered food: Soybeans; corn; cotton

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Plant Genetic Engineering

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Animal Husbandry
• Transgenic animals model advancements in DNA technology in their
development. The mechanism for creating one can be described in
three steps:
• Healthy egg cells are removed from a female of the host animal and
fertilized in the laboratory.
• The desired gene from another species is identified, isolated, and
cloned.
• The cloned genes are injected directly into the eggs, which are then
surgically implanted in the host female, where the embryo undergoes
a normal development process.
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Animal Husbandry: Illustration

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Ethical considerations: Pros
• Crops: Taste better; less ripening time; higher nutrient content;
improved resistance to a variety of factors; new products & growing
techniques.
• Animals: Increased resistance, productivity, hardiness etc.; better
yields of meat etc.; improved animal health.
• Environment: Environmentally friendly herbicides etc.; conservation;
better waste management; more efficient processing.
• Society: More food for growing populations

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Ethical considerations: Cons
• Safety issues

• Access and intellectual property

• Ethics: Tampering with nature; stress to animals etc

• Labeling: Not mandatory in some countries

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