Download Chapter 23 (Part 1)

Chapter 23 (Part 1)
Recombinant DNA Technology
Recombinant DNA
Technology
• Methods for isolating, manipulating,
and amplifying identifiable DNA
sequences.
• Allows us to study the structure
and function of individual genes.
• Allows for the directed genetic
manipulation of organism (modify
gene function, insert novel genes)
Cloning
• Clone: a collection of molecules or cells, all
identical to an original molecule or cell
• To "clone a gene" is to make many copies of
it - for example, in a population of bacteria
• Gene can be an exact copy of a natural gene
• Gene can be an altered version of a natural
gene
• Recombinant DNA technology makes it
possible
• Allows for in vitro manipulation of a
individual gene
Tools Needed for Cloning
(Think of it as a cutting and pasting process)
• cDNA or genomic library (source of DNA
to cut)
• Plasmid (where you want to paste it)
• Restriction enzymes (scissors)
• DNA ligase (paste)
• E. coli (biological machine needed to
amplify DNA)
Plasmids
• Naturally occurring extrachromosomal DNA
• Self replicating circular double stranded
DNA molecules that have their own origin
of replication
• Usually present in multiple copies per cell
• Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
• Artificial plasmids can be constructed by
linking new DNA fragments to the sticky
ends of plasmid
Cloning Vector
Required features
1. Origin of replication
2. Selectable marker
3. Screenable marker
for recombinant
molecules
4. Cloning sites
Restriction Enzymes
• Bacteria protect themselves from attack by
viruses and other bacteria using a
restriction/modification system.
• Allows bacteria to recognize and destroy
foreign DNA
• Bacteria contain DNA methylases that modify
their chromosomal DNA at specific sequences.
• Also contain restriction endonucleases that
recognize and cleave these same sequences
when they are not methylated
Restriction Modification System
AAGATGCGAATTCGTACA
DNA methylase
AAGATGCGAATTCGTACA
DNA methylase
*
*
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
Restriction endonuclease
Restriction endonuclease
*
*
AAGATGCGAATTCGTACA
AAGATGCG
AATTCGTACA
Restriction Enzymes
• Type I – Contain methylase and
endonulcease fuctions. Require ATP for
hydrolysis and S-adenosylmethionine for
methylation
• Type II – contain only endonulcease
function,. Does not require ATP for
hydrolysis.
• Both types recognize palindrome
sequences (sequences that read the same
if read forward or backwards – e.g.
“BOB” or “DEED”
Type II Restriction
Enzymes
• Names use 3-letter italicized code:
• 1st letter - genus; 2nd,3rd species
• Following letter denotes strain
• EcoRI is the first restriction
enzyme found in the R strain of E.
coli
5’ ATGCGAATTCCGGTT 3’
3’ TACGCTTAAGGCCTT 5’
EcoR1
Sticky-end cutter
5’-ATGCG-3’
5’-AATTCCGGTT-3’
3’-TACGCTTAA-5’
3’-GGCCTT-5’
5’ ATGCGATATCCGGTT 3’
3’ TACGCTATAGGCCTT 5’
Blunt-end cutter
EcoRV
5’-ATGCGAT-3’
3’-TACGCTA-5’
5’-ATCCGGTT-3’
3’-TAGGCCTT-5’
Restriction Enzymes
• Restriction enzymes can recognize specific 4 base, 6
base, 8 base sequences.
• The probability that a given piece of DNA will contain
a specific restriction site is = n4
• n = the number of bases in the restriction site
• So for a 6 base cutter (64), you would expect to find
your site every ~1300 base pairs. So in a 10,000 bp
fragment there is likely to by 7 or 8 restriction sites
corresponding to your enzyme.
• You can characterize DNA fragments using gel
electrophoresis
T4 DNA
Ligase
Transformation
• All of the previous steps were
performed in vitro.
• We have generated a very
small amount of a recombinant
plasmid
• Need to amplify in bacteria to
get enough to work with.
• Transformation – process to
mobilize DNA into bacterial
host
• Select for transformed
bacteria on specific antibiotic
that corresponds to the
antibiotic resistance gene
present on the plasmid
How to produce a
recombinant protein
0.1 to 1%
of cellular
protein
10 to 70%
of cellular
protein
Cloning a gene from a DNA libraries
• Any particular gene may represent a
tiny, tiny fraction of the DNA in a
given cell
• Can't isolate it directly
• Trick is to find the fragment or
fragments in the library that contains
the desired gene
cDNA
cDNA
Library
cDNA
Library Screening
• DNA probe hydridization
• Requires that you know the protein or amino acid
sequence of the gene of interest.
• Need to denature (make single stranded) and
immobilize the DNA from each clone of the library to
a filter (nitrocellulose or nylon)
• Make a labeled single stranded DNA/RNA probe (can
use radioactive of fluorescent analogous of specific
nucleotide triphosphates)
• Labeled single stranded DNA/RNA fragments will
base pair (hydridize) with the target DNA on the
filter
• Identify clones that are labeled.
DNA
hydridization
screening for
specific gene
•Requires that you know
something about the gene
sequence
•Can get sequence
information form purified
protein
Now that we have the gene,
what do we do with it?
• We could use it make a lot of
protein in a microbial protein
expression system
• We could use it to genetically
manipulate organisms
• We could use it as a diagnostic tool
Why use recombinant
Proteins?
• Proteins are often only available in
small amounts in a given tissue
• Tissue sources may not be readily
available
• It is time consuming and expensive
to purify protein from tissues
• It is difficult to obtain absolutely
pure protein
Insulin
• Was first purified from human
pancreas from cadavers and then
from pig pancreas.
• Genentec expressed insulin gene in
microbial host
• Can grow microbes in large
fermenters to produce unlimited
supply of insulin.
Product name
Protein type
Application
Company
Adagen (Adenosine
deaminase )
An enzyme
Severe combined
immunodeficiency
disease (SCID)
Enzon
Genotropin
(Recombinant
growth hormone)
A hormone
Growth hormone
deficiency (GHD) in
children
Pharmacia & Upjohn
Humalog
(Recombinant human
insulin)
A hormone
Diabetes
Eli Lilly
Nabi-HB (AntiHepatitis B)
An antibody
Hepatitis-B
Nabi
Novo Seven
(Recombinant
coagulation factor
VIIa)
A modified factor
Hemophillia patients
with inhibitors
Novo Nordisk
Ontak (Diphtheria
toxin-interleukin-2)
A fusion protein
Cutaneous T-cell
lymphoma (CTCL)
Ligand
Pharmaceuticals
Roferon-A
(Recombinant
interferon alfa-2a)
A modifier
Hairy cell leukemia
or AIDS-related
Kaposi's sarcoma
Hoffmann-La Roche
Recombinant proteins are
also important to research
• For enzyme analysis need pure
protein
• For structural analysis need lots
(milligram amounts) of very pure
protein
• Need pure proteins to make
diagnostic tools such as antibodies
Genetic Modification of
Higher Organisms
• Can introduce gene into animals and
plants
• These modified organism are powerful
research tools to study the effect of a
specific gene product on metabolism,
development etc….
• Has also been used to develop improved
agricultural products
Genetically Engineered Salmon
Is Bigger Better?
http://www.agwest.sk.ca/sabic_index_tp.shtml
Plant Genetic Engineering
Improved Agricultural Production
A. Herbicide Resistance
B. Pest Resistance
Improved Nutrition
A. Vitamins - Golden Rice, Vitamin E
B. Increase essential Amino Acid Content
Chemical Synthesis
A. Bio-plastics
B. Bio-diesel
C. Lubricants/detergents
D. Rubber
GMO Concerns
• Ecological Concern
• Potential Food Allergens
• Antibiotic Resistance
GMO Benefits
• Lower application of herbicides and
pesticides
• Creation of foods with increased
nutrition
• Creation of bio-based alternative to
petroleum based products
http://www.colostate.edu/programs/lifesciences/TransgenicCrops/