Biotechnology

Chapter 10

Biotechnology
Biotechnology was used to create the pGLO cloning
plasmid (chapter 9 notes)
A jelly fish gene was combined with bacterial DNA ultimately
resulting in fluorescent bacteria

Biotechnology
Biotechnology is possible due to
The central dogma
All living organisms genetic information is encoded in DNA which is
transcribed and translated to create a protein resulting in a particular
trait
The DNA and process is so similar that a gene from one organism can be
used by other organisms
Transcriptio
n

Translation

Biotechnology
Biotechnology is possible due to
Knowing the structure of DNA as well as the enzymes involved
in its function
Restriction enzymes
Each type of restriction enzyme cuts DNA at very specific nucleotide
sequences
EcoR1 cuts at the sequence GAATTC

The cuts are staggered leaving sticky ends

Each sticky end can base-pair with a complementary sticky end on a different
fragment of DNA

Biotechnology
Biotechnology is possible due to
Knowing the structure of DNA as well as the enzymes involved
in its function
Complementary base pairing or hybridization
Any nucleotide sequence that is complementary to another sequence can
bind forming double-stranded DNA

Biotechnology
Biotechnology is possible due to
Knowing the structure of DNA as well as the enzymes involved
in its function
Ligase
The enzyme used to bind the phosphate-sugar backbone between
nucleotides

The combination of restriction enzymes and ligase allows for cutting and
pasting of any two pieces of DNA
Forms recombinant DNA

DNA Cloning
A set of methods that uses living cells to
make many identical copies of a DNA
fragment
Allows manipulation and analysis of a gene from
one organism in a second organism
Example using pGLO

DNA Cloning
Example using pGLO
Jellyfish (Aequorea victoria) express the
trait of fluorescence
Gene transcribed to mRNA translated to
a polypeptide trait is expressed

Extract jellyfish DNA

Same basic method as with the strawberry
DNA extraction lab
The DNA sample will contain multiple copies
of all the chromosomes with all of the various
genes for a jellyfish

Cut the jellyfish DNA with a specific

restriction enzyme

DNA Cloning
Example using pGLO
Plasmids are manipulated to work as cloning
vectors
Plasmids are small circles of DNA with just a few genes
Non-essential for survival and not part of the major
bacterial chromosome
Plasmids are copied and distributed to daughter cells when
Bacterium
Plasmid
the bacterium divides
Chromosome

DNA Cloning
Example using pGLO
Plasmids are manipulated to work as cloning vectors
Addition of restriction enzyme sites
To create recombinant plasmids with foreign DNA fragments

Addition of bacterial genes

To help select bacteria that take up cloning plasmids
Antibiotic resistance

DNA Cloning
Example using pGLO
Cut the cloning plasmid with the same specific
restriction enzyme used on the jelly fish DNA
Creates complimentary sticky ends

DNA Cloning
Example using pGLO
The cut plasmid and jelly fish DNA fragments are
mixed with DNA ligase
Because the plasmid and fragments have the same
sticky ends, they will base pair
DNA ligase glues the back-bone of the two DNAs
together forming recombinant plasmids
Some plasmids will close back up without any foreign
DNA

DNA Cloning
Example using pGLO
The recombinant plasmids are mixed with host bacterial cells
A few bacteria take up recombinants plasmids
Referred to as transformed cells

DNA Cloning
Example using pGLO
The bacteria are plated on nutrient agar with ampicillin (an
antibiotic)
Only transformed cells containing the plasmid will survive
Due to the antibiotic resistance gene in the plasmid

X
X

DNA Cloning
Example using pGLO
Each surviving, transformed bacterium will divide multiple
times creating a colony of identical bacterial cells or clones
Each clone contains a copy of the vector with the foreign DNA

Selection of clones
Look for the trait coded by the foreign gene
pGLO = fluorescence

Recombinant DNA cloning

DNA Cloning
Example using pGLO
The only changes in the bacteria are
Fluorescence
Ampicillin resistance

All other traits remain the same

Would the bacteria become dangerous in any way?
It is a genetically modified organism (GMO)

Genetically Modified Organisms

Is rice harmful?
Are carrots harmful?
Is it harmful to mix rice and carrots?

Genetically Modified Organisms

Are various strains of rice harmful?
Each has a set of genes that code for the traits to make rice
Each has a few different genes that result in variations of traits

Genetically Modified Organisms

Would taking a gene from carrots and putting it in rice
make the rice harmful?
The carrot gene would be transcribed and translated to produce
the carrot protein in the rice
The carrot protein is harmless in carrots and would be harmless
in rice
Would genetically modified rice be harmful?

Genetically Modified Organisms

Almost all of the food we eat is derived from genetically
modified organisms
Genetic changes have been occurring in nature for billions of years
Mutations, crossing over, etc.

Humans have directed genetic changes for thousands of years using

artificial selection
New/improved crop plants
Corn (originated from teosinte)
Sweeter oranges, tangelos, different varieties

Improved farm animals

Meatier turkeys, creamier milk producing cows

New breeds of pets

Dogs, cats, birds

Genetically Modified Organisms

Genetically modified organisms are not inherently
unhealthy or bad for us
Design a well controlled good experiment
Control group and experimental group
Length (years, multiple generations)
Difficult

Genetically Modified Organisms

Genetically modified organisms are not inherently
unhealthy or bad for us
Information is available

(Journal of Animal Science, Eenennaam and Young, 2014)

29 years of livestock productivity and health data

Includes animals fed pre-GMO and post-GMO feed
Multiple generations and types of animals included
More than 100 billion animals

No indications of any unusual trends in the health of animals since 1996

when GMO crops were first harvested
Zero extraordinary impact

Genetically Modified Organisms

Genetically modified organisms are not inherently
unhealthy or bad for us
Beware information relying on
Anecdotal stories
Self-published studies
Scare-tactics writing style
Claim about grotesque cancerous tumorsthe kind no farmer would miss
If GMO feed caused unusual problems among livestock farmers would have
noticed--Dead and sick animals would literally litter farms around the world.
Sterility would mean fewer numbers of livestock. Etc.
Drive around, ask farmers

Genetically Modified Organisms

Genetically modified organisms are not inherently
unhealthy or bad for us
There are legitimate concerns
Policies, lobbyists, marketing
Agricultural practices
Monocultures
Cross pollination

Genetically Modified Organisms

http://
mediasite.video.ufl.edu/Mediasite/Play/4789e14d844c4a74ab
a2d7df829b132a1d

Other DNA Techniques

cDNA
Extract mRNA from only those cells producing the gene
product
Since only about 10% of a cells genes are expressed, this greatly
increases the odds of having a DNA fragment with the gene of
interest
Jelly fish: cells that fluoresce

mRNA is reverse transcribed into a single strand of DNA

Using the enzyme reverse transcriptase

Replicate the new single strands of DNA to form doublestranded fragments of DNA
Using the enzyme DNA polymerase

Extra benefit: the introns of eukaryotic genes have been

removed

Other DNA Techniques

PCR
Isolates and mass-produces a particular DNA fragment

PCR Method
Start with a sample of DNA that will have the target sequence
Could be DNA from different clones, one sperm, a hair left at a crime
scene, or a mummy
Any sample that has DNA in it can be used

Other DNA Techniques

PCR Method
Mix the sample of DNA with
Heat tolerant DNA polymerase (from Thermus aquaticus)
Free nucleotides
Primers
Short single strands of DNA (10-30 bases long)
Designed to hybridize (complementary base-pair) at either end of the DNA
sequence to be amplified

Other DNA Techniques

PCR Method
Expose the mixture to repeated cycles of high and low
temperature
Heat: double strands of DNA unwind and separate
Cool: complementary base pairing occurs between single-stranded
DNA and primers
DNA polymerase synthesizes the target DNA wherever the primers bind

Repeat the cycle

After every cycle the number of copies of target DNA can double
Ultimately amplifying about a billion fold

PCR

Other DNA Techniques

DNA Sequencing
Process to determine the ATCG
sequence of any given segment of
DNA
Human genome has been sequenced

Other DNA Techniques

DNA Gel Electrophoresis
The gel
Agarose gels are viscous jello-like material
DNA fragments can be forced to travel through the gel by running an
electric current through the gel

DNA
DNA is negatively charged (due to the phosphate groups)
Will migrate toward the positive electrode
The longer the fragment of DNA, the slower it migrates
The shorter the fragment of DNA, the faster it migrates

Other DNA Techniques

DNA Gel Electrophoresis
The process
Liquefied agarose is poured into a tray and a comb is placed at one end
to create wells
Once the gel has set up it is placed in an electrophoresis chamber with a
buffer solution

Other DNA Techniques

DNA Gel Electrophoresis
The process
DNA samples are added to each well
Samples consist of thousands of pieces or
fragments of DNA
All the fragments the same size will migrate
together to form a band of DNA in the gel
Multiple bands form if there are several different
lengths of DNA fragments

Other DNA Techniques

DNA Gel Electrophoresis
The process
The apparatus is connected to a power source creating an electric
current though the gel
DNA migrates toward the positive electrode
Fragments separate based on size

Gel electrophoresis

Other DNA Techniques

DNA Fingerprinting
Each individual has unique DNA sequences
(only about 1%, the other 99% is the same)

Some of the unique sequences are called short tandem repeats

Many copies of the same 2-10 base pair sequences
Positioned one after the other along the length of a chromosome
TTTTCTTTTCTTTTCTTTTC
CGGCGGCGGCGGCGGCGG

The number of short tandem repeats differs from individual to individual

From 5 up to 50 times

Other DNA Techniques

DNA Fingerprinting
Process
PCR is used to amplify a region of a chromosome known to have tandem
repeats (several regions are actually used)
The size of fragments differs among individuals because the number of
tandem repeats also differs
An individual with only 5 repeats will generate short fragments
An individual with up to 50 repeats will generate long fragments
The Fragments are separated by gel electrophoresis

Other DNA Techniques

DNA Fingerprinting
The resulting banding patterns on the gel are an individuals
DNA fingerprint
If three regions are tested
1 in 1X1018 chance of two people having the same banding pattern
(except for identical twins)
1X1018 is far more than the number of people on Earth

Boyfriend
Control DNA
Control DNA
Size Reference

Size Reference

Size Reference
Control DNA
Size Reference
Victim
Suspect 1
Suspect 2
Female Cells
Semen

Evidence from
Crime Scene

Other DNA Techniques

DNA Hybridization
Complementary base pairing or hybridization
Can be used to identify or located specific
sequences of DNA
PCR primers work based on hybridization

Other DNA Techniques

DNA Hybridization
Complementary base pairing or hybridization
Analysis of the human genome SNP (single-nucleotide polymorphism)
Similar to DNA fingerprinting

Practical Applications of DNA technology

Medical
Produce medicines quickly and inexpensively
Diagnosis and treatment of genetic diseases
Gene therapy

Forensic Evidence
Determine guilt
Determine paternity
Determine identity

Environmental Cleanup
Extract heavy metals
Clean up oil spills
Degrade chlorinated hydrocarbons in wastewater treatment plants

Agricultural Applications

Genetically Modified Organisms

Research and information
http://factsaboutgmos.org/
http://www.scientificamerican.com/article/labels-for-gmo-foods-ar
e-a-bad-idea
/
http://geneticliteracyproject.org/2014/09/10/19-year-study-of-tri
llions-of-meals-shows-ge-crops-do-not-harm-food-producing-animals
-humans
/
http://geneticliteracyproject.org/2015/03/03/humans-have-been-pl
aying-god-with-farm-crops-for-1000s-of-years-and-thats-okay/?
relatedposts_hit=1&relatedposts_origin=268632&relatedposts_po
sition=2
https://gmoanswers.com/studies/october-get-know-gmos-mont
h#./october-get-know-gmos-month?&_suid=143352919549003727