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BIOTECHNOLOGY
IB BIOLOGY
Recombinant DNA
 Defined:
 DNA that contains DNA from other
sources
 Transfer from one part of DNA
molecule to another
 Transfer from one c’some to
another
 Transfer from one organism to
another!
Can occur artificially…
RECOMBINANT DNA
TECHNOLOGY
What is Recombinant DNA
Technology?
 1974
 Overview:
 Select and isolate specific sequence of
DNA in organism of choice (ie: human
gene)
 Incorporate selected DNA sequence into
DNA of a vector organism
 Grow and amplify resulting recombinant
DNA in a host that reproduces quickly
Overview
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
The Specifics:
Requires use of
RESTRICTION ENZYMES
naturally occurring in bacteria
(1960’s)
natural protection against viral
infection
How are Restriction Enzymes
used in Recombinant DNA
Technology?
THREE Parts:
1. Occurs in organism of choice
2. Occurs in vector organism
3. Occurs in host organism
Part I:
A. Restriction enzymes obtained
from bacteria (ex: EcoRI) and
inserted into DNA of organism of
choice!
B. Restriction Enzyme specifically
cuts DNA of selected organism
at specific recognition sequences
of nucleotides.
C. Restriction Enzyme cuts across DNA
double helix, producing restriction
fragments with staggered ends
extending beyond complementary
strand (“sticky ends”)
EcoRI
LE 20-3
Restriction site
DNA 5
3
3
5
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
Sticky end
DNA fragment from another
source is added. Base pairing
of sticky ends produces
various combinations.
Using a
restriction
enzyme and
DNA ligase
to make
recombinant
DNA
Fragment from different
DNA molecule cut by the
same restriction enzyme
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule
Part II:
THE MOST COMMON
RECOMBINANT DNA VECTORS
ARE PLASMIDS AND
BACTERIOPHAGES!
D. Treat plasmid with same restriction
enzyme as was used to make the
DNA restriction fragment in selected
organism. (produces same sticky
ends as carried by fragment)!
E. Mix the two strands of DNA (selected
organism and vector) allowing for
base pairing at sticky ends.
F. Use DNA ligase to stabilize
attachment
G. Recombinant Plasmid is now
formed. CONGRATULATIONS.
Part III:
IN ORDER TO BE EFFECTIVE, HOST
ORGANISM SELECTED MUST BE
CAPABLE OF REPRODUCING AT A
RAPID RATE!
why?
H. Recombinant plasmid is
introduced into a bacterial host
organism by transformation.
I. Host organism reproduces,
thereby multiplying the
recombinant DNA plasmid!
Genetically engineered Human insulin
 http://www.abpischools.org.uk/res/co
ResourceImport/modules/hormones/e
n-flash/geneticeng.cfm
Genetic Engineering Techniques
Definition:
Technology that uses genetic and
recombinant DNA methods to devise
new combinations of genes to
produce improved pharmaceutical
and agricultural products.
Genes isolated from one
organism can be modified
and expressed in another
organism.
(E. coli, transgenic plants,
transgenic animals)
Transgenic Plants
Transgenic Bacteria
Genetically Modified
Organisms
C L O N I N G
 Process which genetically
identical cells and/or individuals
are created.
 Gene Cloning:
recombinant DNA injected into
highly reproductive host
organism (E. coli)
[see notes]
 Organismal Cloning:
where the controversy arises…
How do you clone an
individual?
1. Haploid nucleus in ovum is removed
from organism A and replaced with
diploid nucleus from somatic cell
taken from organism B.
2. Ovum (with diploid nucleus) is
reinserted into uterus of Organism C
(surrogate mother), where it will
develop to term.
3. Organism born from altered ovum is
genetically identical to organism A.
Successful Vertebrate Cloning
Experiments
 Amphibians (1970’s)
 Mammals






Sheep (DOLLY)
Mice
Cattle
Pigs
Cats (CC)
Humans????
Dolly
July 5, 1996 – Feb 14, 2003
RIP
Polymerase Chain Reaction
PCR
Developed 1983
Kerry Mullis
What does PCR do?
Amplifies small sample of
DNA millions of times in a
few hours rather than
cloning an entire cell!
Can generate ~1million
copies of DNA for every 20
cycles of PCR.
The PCR Process:
1. DNA polymerase is used to
replicate the targeted DNA
sequence.
Replicate  heat to
separateReplicate
Uses for PCR
 Fossil Analysis
 Criminal Cases
Gel Electrophoresis
 DNA fragments of different lengths
are separated as they diffuse through
a gel under the influence of an
electrical field.
 DNA is negatively charged
(phosphate groups)
they
move toward the positive electrode!
 Shorter fragments move further
through the gel than longer
fragments! (WHY?)
USES of Gel Electrophoresis
 Criminal Court Cases
 Determination of evolutionary
relationships between closely related
species.
DNA Fingerprinting
 Comparing the DNA from a
known individual to the DNA
from an unknown sample
 Uses: Criminology
Paternity Tests
How does DNA Fingerprinting
Work?
 When Restriction Fragments between
individuals of the same species are
compared, the fragments differ in length
due to polymorphisms (slight differences in
sequences).
 Fragments are known as RFLP’s (Restriction
Fragment Length Polymorphisms)
 By using gel electrophoresis, RFLP’s from
different individuals can be compared to
determine relatedness!
 GENETIC SCREENING
 GENE THERAPY