The DNA Toolbox

• DNA sequencing has depended on advances in

technology, starting with making recombinant DNA

• a recombinant DNA molecule is one that contains

some foreign DNA "inserted" into it, so it recombines.

• nucleotide sequences from two different sources,

often two species, are combined in vitro into the
same DNA molecule
• Methods for making recombinant DNA are central
to genetic engineering, the direct manipulation of
genes for practical purposes

• DNA technology has revolutionized biotechnology,

the manipulation of organisms or their genetic
components to make useful products

• An example of DNA technology is the microarray, a

measurement of gene expression of thousands of
different genes
A DNA microarray allows scientists to perform an
experiment on thousands of genes at the same
time.
 DNA microarray
• A DNA microarray (also commonly known
as DNA chip or biochip) is a collection of
microscopic DNA spots attached to a solid surface.

• Microarray analysis involves breaking open a cell,

isolating its genetic contents, identifying all the genes
that are turned on in that particular cell, and
generating a list of those genes.
 The practical applications of DNA
technology affect our lives in many ways

• Many fields benefit from DNA technology

and genetic engineering
 Medical Applications

• One benefit of DNA technology is identification of

human genes in which mutation plays a role in
genetic diseases
 Diagnosis of Diseases
• Scientists can diagnose many human genetic
disorders by using PCR and primers corresponding
to cloned disease genes, then sequencing the
amplified product to look for the disease-causing
mutation

• Genetic disorders can also be tested for using

genetic markers that are linked to the disease-
causing allele
 Amplifying DNA in Vitro: The Polymerase
Chain Reaction (PCR)

• The polymerase chain reaction, PCR, can produce

many copies of a specific target segment of DNA

• A three-step cycle—heating, cooling, and

replication—brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules
• Single nucleotide polymorphisms (SNPs) are useful
genetic markers

• These are single base-pair sites that vary in a

population

• When a restriction enzyme is added, SNPs result in

DNA fragments with different lengths, or restriction
fragment length polymorphism (RFLP)
Fig. 20-21

Single nucleotide polymorphisms (SNPs) as

genetic markers for disease-causing alleles

DNA
T
Normal allele
SNP

C
Disease-causing
allele
 Human Gene Therapy
• Gene therapy is an experimental technique that uses
genes to treat or prevent disease

• Gene therapy is the alteration of an afflicted

individual’s genes

• Gene therapy holds great potential for treating

disorders traceable to a single defective gene
 Approaches to gene therapy
• 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.
 Human Gene Therapy
• Vectors are used for delivery of genes into specific
types of cells, for example bone marrow

• Gene therapy raises ethical questions, such as

whether human germ-line cells should be treated
to correct the defect in future generations
All viruses bind to their hosts and introduce their genetic material into
the host cell as part of their replication cycle
 Human Gene Therapy
• Gene therapy is a promising treatment option for a
number of diseases (including inherited disorders,
some types of cancer, and certain viral infections),

• Gene therapy trials using retroviral vectors to treat X-

linked severe combined immunodeficiency (X-SCID)
represent the most successful application of gene
therapy to date.
 Human Gene Therapy
• The genetic material in retroviruses is in the form
of RNA molecules, while the genetic material of their
hosts is in the form of DNA.

• When a retrovirus infects a host cell, it will introduce

its RNA together with some enzymes, namely reverse
transcriptase and integrase, into the cell.
 Human Gene Therapy

• The technique remains risky and is still under study

to make sure that it will be safe and effective.

• Gene therapy is currently being tested only for

diseases that have no other cures.
GMOs and Genetic Engineering
Genetically Modified Foods
 Genetically Modified Organisms
• A genetically modified organism (GMO) is
any organism whose genetic material has
been altered using genetic engineering
techniques (i.e., a genetically engineered
organism).
 What are GM’s?
Other terms that mean the same thing:
• Genetically engineered
• Transgenic
• Recombinant DNA (rDNA) technology
How does this differ from Mendel and his peas?
GM vs. Selective breading
Selective breading
-slow
-imprecise
-modification of genes that naturally occur in the
organism
GM
-very fast
-precise
-can introduce genes into an organism that would not
occur naturally!
 Why do it?
• Rice- not high in essential nutrients

Modification:
+ daffodil genes and a bacterium = beta-carotene
content drastically increased
+ genes from a french bean = double the iron
content.

• Tomatoes- Introduce genes to increase shelf

life.
How is this done?: Transgenic tomatoes
 Pharmaceutical Products
• Advances in DNA technology and genetic research
are important to the development of new drugs to
treat diseases
 Synthesis of Small Molecules for Use as Drugs

• The drug imatinib is a small molecule that inhibits

overexpression of a specific leukemia-causing
receptor

• Pharmaceutical products that are proteins can be

synthesized on a large scale
 Protein Production in Cell Cultures

• Host cells in culture can be engineered to secrete a

protein as it is made

• This is useful for the production of insulin, human

growth hormones, and vaccines
 Protein Production by “Pharm” Animals and Plants

• Transgenic animals are made by introducing genes

from one species into the genome of another
animal

• Transgenic animals are pharmaceutical “factories,”

producers of large amounts of otherwise rare
substances for medical use

• “Pharm” plants are also being developed to make

human proteins for medical use
Goats as “pharm” animals
 Environmental Cleanup
• Genetic engineering can be used to modify the
metabolism of microorganisms

• Some modified microorganisms can be used to

extract minerals from the environment or degrade
potentially toxic waste materials

• Biofuels make use of crops such as corn, soybeans,

and cassava to replace fossil fuels
 Agricultural Applications
• DNA technology is being used to improve
agricultural productivity and food quality

• Agricultural scientists have endowed a number of

crop plants with genes for desirable traits
 Animal Husbandry
• Genetic engineering of transgenic animals speeds
up the selective breeding process

• Beneficial genes can be transferred between

varieties or species
• Most public concern about possible hazards centers
on genetically modified (GM) organisms used as
food

• Some are concerned about the creation of “super

weeds” from the transfer of genes from GM crops
to their wild relatives
• As biotechnology continues to change, so does its
use in agriculture, industry, and medicine

• National agencies and international organizations

strive to set guidelines for safe and ethical practices
in the use of biotechnology
 Forensic Evidence and Genetic Profiles

• An individual’s unique DNA sequence, or genetic

profile, can be obtained by analysis of tissue or
body fluids
• Genetic profiles can be used to provide evidence in
criminal and paternity cases and to identify human
remains
• Genetic profiles can be analyzed using RFLP analysis
by Southern blotting
• Even more sensitive is the use of genetic markers
called short tandem repeats (STRs), which are
variations in the number of repeats of specific DNA
sequences

• PCR and gel electrophoresis are used to amplify

and then identify STRs of different lengths

• The probability that two people who are not

identical twins have the same STR markers is
exceptionally small
This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.

Source of STR STR STR

sample marker 1 marker 2 marker 3

Semen on victim 17, 19 13, 16 12, 12

Earl Washington 16, 18 14, 15 11, 12

Kenneth Tinsley 17, 19 13, 16 12, 12

(b) These and other STR data exonerated Washington and

led Tinsley to plead guilty to the murder.
 Benefits of Genetic Engineering
and Modifying
1. Higher yielding crops, more efficient use of land
2. Can save money and promote higher profits
3. Longer shelf life, less waste
Example// Tomatoes from genetically
modified seeds stay fresh
longer.
4. Enhanced taste and quality
5. Reduced maturation time
 Benefits of Genetic Engineering and Modifying
6. Increased and improved nutrients and stress tolerance

- A single gene genetically engineered into

cauliflower can increase production of beta-carotene
100 times.

- A gene can be implanted into a soybean upgrading

the soy protein to a quality equal to that of milk.

- Corn can be modified to contain its two limiting

amino acids, lysine or tryptophan
 Benefits of Genetic Engineering and Modifying
7. Improved resistance to disease or illness
- Foods can be enhanced with phytochemicals that help
maintain health and reduce the risks of chronic disease.

8. Improved crop resistance to disease, pests, weeds and

herbicides

9. New products and growing techniques

- “Individuals allergic to milk may be able to buy milk that has
been treated with the lactase enzyme” (Whiney, 2002).

- Creating decaffeinated coffee beans are in a process of

research.
 Benefits of Genetic Engineering and Modifying

• Society
– Increased food
security for growing
populations and
growth challenges
(Human Genome Project Information (2003),
http://www.ornl.gov/sci/techresources/Human
_Genome/elsi/gmfood.shtml)
Who Uses this technology
The Countries that Grow 99% of the
World's Transgenic Crops

7% 1%

USA
23%
Argentina
Canada

69% China
 Risks associated with Genetic Modification

1. Safety
– Potential human health implications.
– Potential environmental impact.
• Out-crossing
– Inevitable out-crossing of transgenic plants with naturally occurring
ones.
– Creation of super-weeds

– Creation of biological weapons.

2. Access and Intellectual Property

– Domination of world food production by a few companies
and developing countries.
Risks associated with Genetic Modification
– cont.
3. Ethics
– “Playing God”
– Tampering with nature by mixing genes among species.

4. Labeling
– Not mandatory in some countries (e.g., Canada and the United States).
– Mixing GM crops with non-GM confounds labeling attempts.

5. Society
– New advances may be skewed to the interests of rich countries.
(Human Genome Project Information (2003), http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood.shtml)
 Risks with GM

Biodiversity
• Addition of Bt gene into plants including corn, potatoes
and cotton to increase resistance to plants

• Bt gene obtained from Bacillus thuringiensis (a soil

bacterium that produces a natural insecticide)

• Problem: plants producing Bt toxin are releasing toxin in

pollen

Draper, D. (2002). Our Environment: A Canadian Perspective 2nd Ed. Scarborough: Thompson Canada Lmt.
• Pollen from a Bt plant was dusted on to milkweed:
- only 56% of young monarch butterfly larvae lived

- whereas pollen from organic plants dusted on the

milkweed produced a survival rate of 100%.

Approximately half of the monarch butterfly

population live in the “corn belt” of the USA

- this new gene could have serious repercussions for

this organism
 Safety and Ethical Questions Raised by DNA
Technology

• Potential benefits of genetic engineering must be

weighed against potential hazards of creating
harmful products or procedures

• Guidelines are in place in the United States and

other countries to ensure safe practices for
recombinant DNA technology
Genetic Modification

or ?
What is crispr
https://www.youtube.com/watch?v=MnYppmstxIs
• CRISPR Cas9 - A Brief Introduction
• https://www.youtube.com/watch?v=1aJxXWk
E3Ek

• What is crispr
• https://www.youtube.com/watch?v=MnYppm
stxIs
The new tool in gene editing
revolution
https://pbs.twimg.com/media/Ejt54GdWoAINXz3?format=jpg&name=small