Transcript Biotech Overview

Recombinant DNA
and Genetic
Engineering
Chapter 16
Definitions
Recombinant DNA technology:
techniques & tools used to analyze genes
 Genetic Engineering: uses the above
technology to isolate & modify genes in
organisms or even to insert new genes

Genetic Engineering
Genes are isolated, modified, and inserted
into an organism
 Made possible by recombinant technology

 Cut
DNA up and recombine pieces
 Amplify
modified pieces
Restriction Enzymes
Molecular scissors that cut DNA at a
specific nucleotide sequence
 Over 200 different restriction enzymes are
known, each isolated from bacteria and
able to cut DNA in a unique manner

Restriction
enzymes
Recombinant DNA Technology
“Cutting and
Pasting”
Enzymes:
 Restriction
enzymes =
“cut”
 Ligase =
“paste”

DNA (Gene) cloning
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Want to study or isolate a particular gene
Need to get many copies (amplification) of the
gene so it can be studied adequately
Most organisms only have one or two copies of
any gene per cell, so we need a way to amplify
copies of that gene
Do that via cloning into a vector
This allows scientists to make additional copies
of the gene using bacteria
Using Plasmids

Plasmid is small circle of bacterial DNA

Foreign DNA can be inserted into
plasmid
 Forms
recombinant plasmids
 Plasmid
 Can
is a cloning vector
deliver DNA into another cell
Using
Plasmids
Polymerase Chain Reaction
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A faster method of amplifying or making more
copies of a gene is PCR
PCR requires short pieces of single-stranded
DNA which match up to a regions at the
beginning & end of the gene to be amplified,
called primers
Primers are required as a starting point for the
DNA polymerase, the same enzyme used in
DNA replication
DNA polymerase then makes copy after copy of
the gene. In a couple of hours more copies can
be made by PCR than are made using bacteria &
cloning vectors
Polymerase Chain Reaction

Sequence to be copied is heated

Primers are added and bind to ends of
single strands

DNA polymerase uses free nucleotides
to create complementary strands

Doubles number of copies of DNA at
each cycle
Polymerase
Chain Reaction
Double-stranded
DNA to copy
DNA heated to
90°– 94°C
Primers added to
base-pair with
ends
Mixture cooled;
base-pairing of
primers and ends
of DNA strands
Stepped Art
Figure 16.6
Page 256
DNA polymerases
assemble new
DNA strands
Polymerase
Chain Reaction
Mixture heated again;
makes all DNA
fragments unwind
Mixture cooled; basepairing between
primers and ends of
single DNA strands
Stepped Art
Figure 16.6
Page 256
DNA polymerase
action again
doubles number of
identical DNA
fragments
Gel Electrophoresis
DNA is placed at one end of a gel
 A current is applied to the gel
 DNA molecules are negatively charged and
move toward positive end of gel
 Smaller molecules move faster than larger
ones
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Gel Electrophoresis
What are these techniques
used for?
Forensic: identifying criminals & victims
 Identifying disease genes in animals &
humans
 Gene Therapy: inserting of new working
copies of genes into humans
 Animal knockouts: turning off of a specific
gene in order to discover its function

DNA Fingerprinting
Engineered Proteins

Bacteria can be used to grow medically
valuable proteins
Insulin,
interferon, blood-clotting factors
Vaccines
Engineered Plants
Cotton plants that display resistance to
herbicide
 Aspen plants that produce less lignin and
more cellulose
 Tobacco plants that produce human
proteins
 Mustard plant cells that produce
biodegradable plastic
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Cloning Dolly
1997 - A sheep cloned from an adult cell
Nucleus
from mammary gland cell was
inserted into enucleated egg
Embryo
implanted into surrogate
mother
Sheep
is genetic replica of animal from
which mammary cell was taken
The Human Genome Initiative
Goal - Map the entire human genome
 Initially thought by many to be a waste of
resources
 Process accelerated when Craig Ventner
used bits of cDNAs as hooks to find genes
 Sequencing was mostly completed ahead
of schedule in early 2001
Ethical Issues
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Who decides what should be
“corrected” through genetic
engineering?
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Should animals be modified to provide
organs for human transplants?
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Should humans be cloned?