Download Biotech 06

Biotechnology
Dolly and
surrogate Mom
Genetically modified rice.
Biotechnology
Biotechnology, defined broadly, is the engineering of organisms for useful
purposes.
Often, biotechnology involves the creation of hybrid genes and their introduction
into organisms in which some or all of the gene is not normally present.
Fourteen month-old genetically engineered (“biotech”) salmon (left) and
standard salmon (right).
Biotechnology
We’ll examine:
Gene cloning for pharmaceutical
production
DNA fingerprinting
Recombinant DNA, Gene Cloning, and Pharmaceutical Production
These are mature and widely utilized biotechnologies.
DNA can be cut at specific sequences using restriction enzymes.
This creates DNA fragments useful for gene cloning.
Restriction Enzymes are Enzymes That Cut DNA Only at
Particular Sequences
Restriction
enzyme
animation
The enzyme EcoRI cutting DNA at its recognition sequence
Different restriction enzymes have different recognition sequences.
This makes it possible to create a wide variety of different gene fragments.
DNAs Cut by a Restriction Enzyme Can be Joined Together
in New Ways
These are recombinant DNAs and they often are made of DNAs from
different organisms.
Plasmids are Used to Replicate a Recombinant DNA
Plasmids are small circles of DNA found in bacteria.
Plasmids replicate independently of the bacterial chromosome.
Pieces of foreign DNA can be added within a plasmid to create a
recombinant plasmid.
Replication often produces 50-100 copies of a recombinant
plasmid in each cell.
Harnessing the Power of Recombinant DNA Technology –
Human Insulin Production by Bacteria
Human Insulin Production by Bacteria
and cut with a restriction enzyme
6) join the plasmid and human fragment
Human Insulin Production by Bacteria
Mix the recombinant plasmid with
bacteria.
Screening bacterial cells to learn which contain the human insulin gene is the
hard part.
Route to the Production by Bacteria of Human Insulin
One cell with the
recombinant plasmid
This is the step when gene cloning takes place.
A fermentor used to grow
recombinant bacteria.
The single recombinant plasmid replicates within a cell.
Then the single cell with many recombinant plasmids produces trillions of
like cells with recombinant plasmid – and the human insulin gene.
Route to the Production by Bacteria of Human Insulin
The final steps are to collect the bacteria, break open the cells, and purify the
insulin protein expressed from the recombinant human insulin gene.
Route to the
Production by
Bacteria of
Human Insulin
Overview of gene
cloning.
Cloning
animation
Reverse transcription: cDNA Cloning
Suppose we need to clone a gene containing lots of
introns. What will happen when the bacterium tries to
express it?
To overcome this problem, we can start with mRNA
isolated from tissues that produce the desired
protein. We then use reverse transcriptase enzyme
(produced by a retrovirus related to HIV) to reverse
transcribe the mRNA into a DNA molecule that now is
free of introns. Now we can ligate "sticky ends" onto
the cDNA and recombine it into a phage or plasmid
vector.
Or: Use Proteins sequence - BLAST website to get
DNA sequence and PCR to make multiple copies of
DNA, the Law, and Many Other Applications –
The Technology of DNA Fingerprinting
A DNA fingerprint used in a murder case.
The defendant stated that the blood on his clothing was his.
What are we looking at? How was it produced?
DNA Fingerprinting Basics
Different individuals carry different alleles.
Most alleles useful for DNA fingerprinting differ on the basis of the
number of repetitive DNA sequences they contain.
DNA Fingerprinting Basics
If DNA is cut with a restriction
enzyme that recognizes sites on either
side of the region that varies, DNA
fragments of different sizes will be
produced.
A DNA fingerprint is made by analyzing
the sizes of DNA fragments produced
from a number of different sites in the
genome that vary in length.
The more common the length variation at
a particular site and the greater the
number the sites analyzed, the more
informative the fingerprint.
A Site With Three Alleles Useful for DNA Fingerprinting
DNA fragments of different size will be produced by a restriction
enzyme that cuts at the points shown by the arrows.
The DNA Fragments Are Separated on the Basis of Size
The technique is gel electrophoresis.
The pattern of DNA bands is compared between each sample loaded on
the gel.
Gel electrophoresis animation
Possible Patterns for a Single “Gene” With Three Alleles
In a standard
DNA
fingerprint,
about a dozen
sites are
analyzed, with
each site
having many
possible
alleles.
A DNA Fingerprint
When many genes are
analyzed, each with many
different alleles, the
chance that two patterns
match by coincidence is
vanishingly small.
Cloning animation
DNA detective
animation
HGP fingerprinting
page
Cloning animation
DNA and the Law
SLT 3/8/05
Some applications of DNA
fingerprinting in the justice system.
Genetically Modified Foods
Many of our crops in the US are genetically modified.
Should they be?
GM Crops are Here Today
Source: Pew Initiative on Food and Biotechnology, August 2004.
Methods for Plant Genetic Engineering are Well-Developed and
Similar to Those for Animals
Golden Rice is Modified to be Provide a Dietary Source of Vitamin A
Golden rice (yellow)
with standard rice
(white).
Worldwide, 7% of children suffer vitamin A deficiency, many of them living in
regions in which rice is a staple of the diet.
Genetically Modified Crops
Genetically Modified Cotton
(contains a bacterial gene for
pest resistance)
Standard Cotton
Current Concerns by Scientists Focus on Environmental, Not
Health, Effects of GM Crops
The jury’s still out on the magnitude of GM crop’s ecological impact, but the
question is debated seriously.
Current Concerns
by Scientists
Focus on
Environmental,
Not Health,
Effects of GM
Crops