Post on 20-Jan-2016
transcript
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 features1. Origin of
replication2. Selectable marker3. 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
AAGATGCGAATTCGTACA* *
AAGATGCGAATTCGTACA* *
DNA methylase
Restriction endonuclease
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
AAGATGCG AATTCGTACA
DNA methylase
Restriction endonuclease
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’
5’-ATGCG-3’ 5’-AATTCCGGTT-3’3’-TACGCTTAA-5’ 3’-GGCCTT-5’
EcoR1
5’ ATGCGATATCCGGTT 3’3’ TACGCTATAGGCCTT 5’
5’-ATGCGAT-3’ 5’-ATCCGGTT-3’3’-TACGCTA-5’ 3’-TAGGCCTT-5’
EcoRV
Sticky-end cutter
Blunt-end cutter
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
10 to 70% of cellular protein
0.1 to 1% 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 (Anti-Hepatitis 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 SalmonIs Bigger Better?
Plant Genetic EngineeringImproved 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/