Recombinant DNA Technology
Recombinant DNA technology is also known as gene cloning
However, it turned out to be safer than expected
- It also spread to industry faster and in more diverse ways than imagined
Figure 8.1 Overview of recombinant DNA technologyBacterial cell
Bacterialchromosome
Plasmid
Gene of interest
DNA containinggene of interest
Isolate plasmid.
Enzymatically cleaveDNA into fragments.
Isolate fragmentwith the gene ofinterest.
Insert gene into plasmid.
Insert plasmid and gene intobacterium.
Culture bacteria.
Harvest copies ofgene to insert intoplants or animals
Harvest proteinscoded by gene
Eliminateundesirablephenotypictraits
Produce vaccines,antibiotics,hormones, orenzymes
Createbeneficialcombinationof traits
DNA Cloning
Goal is to generate large amounts of pure DNA that can be manipulated and studied.
DNA is cloned by the following steps:
1. Isolate DNA from organism (e.g., extract DNA)
2. Cut DNA with restriction enzymes to a desired size.
3. Splice (or ligate) each piece of DNA into a cloning vector to create a recombinant DNA molecule.
Cloning vector = artificial DNA molecule capable of replicating in a host organism (e.g., bacteria).
4. Transform recombinant DNA (cloning vector + DNA fragment) into a host that will replicate and make copies.
5. E. coli is the most common host.
Creating Recombinant DNA Molecules
Manufacturing recombinant DNA requires restriction enzymes that cut donor and recipient DNA at the same sequence
These enzymes cut DNA at sites that are palindromic
The cutting action of many of these enzymes generate single-stranded extensions called “sticky ends”
The Tools of Recombinant DNA Technology
• Restriction Enzymes– Bacterial enzymes that cut DNA molecules only
at restriction sites– Categorized into two groups based on type of
cut• Cuts with sticky ends• Cuts with blunt ends
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MOLECULAR SCISSORS - TYPE II RESTRICTION ENDONUCLEASES
Hamilton Othanel Smith 1968
cohesive ends
SPECIFIC CUT SPECIFIC JOINING (LIGATION)
Ability to join two foreign pieces of DNA together
Step 2-Cut DNA with restriction enzymes
Restriction enzymes recognize specific bases pair sequences in DNA called restriction sites and cleave the DNA by hydrolyzing the phosphodiester bond.
Cut occurs between the 3’ carbon of the first nucleotide and the phosphate of the next nucleotide.
Restriction fragment ends have 5’ phosphates & 3’ hydroxyls.
restriction enzyme
Step 2-Cut DNA with restriction enzymes (cont.)
Most restriction enzymes occur naturally in bacteria.
Protect bacteria against viruses (bacteriophages) by cutting up viral DNA.
Bacteria protects their DNA by modifying possible restriction sites (methylation).
More than 400 restriction enzymes have been isolated.
Names typically begin with 3 italicized letters.
Enzyme SourceEcoRI E. coli RY13HindIII Haemophilus influenzae RdBamHI Bacillus amyloliquefaciens H
Many restriction sites are palindromes of 4-, 6-, or 8-base pairs.
Short restriction site sequences occur more frequently in the genome than longer restriction site sequences.
Their frequency is a function of (1/4)n
Step 2-Cut DNA with restriction enzymes (cont.)
Some restriction enzymes produce blunt ends, whereas others produce sticky (overhanging staggered) ends.
Sticky ends are useful for DNA cloning because complementary sequences anneal and can be joined directly by DNA ligase without using ‘adapters’.
Fig. 8.2
Creating Recombinant DNA Molecules
Another “tool” used is a cloning vector
- Carries DNA from the cells of one species into the cells of another
Commonly used vectors include:
- Plasmids
- Bacteriophages
- Disabled retroviruses
RECOMBINANT DNA TECHNOLGYThe plasmid as DNA ‘vector’ (vehicle)
Possible to insert ‘interesting’ DNA’s into a plasmid using restriction
endonucleases
3
3 Why not clone whole genomes?
Each bacterial colony represents an amplified clone containing a recombinant plasmid harbouring a
distinct region of the genome
i.e. together they represent a ‘Genomic DNA Library’
Also possible to do this using cDNA copies of transcribed mRNAs resulting a ‘cDNA Gene Expression Library’
Bacteriophage Lambdavectors
phage linear DNA genome
Non-essential region that can be substituted by DNA to be cloned (approx 20Kb)
cos cos
Cosmids, phosmids, BACs and YACs to clone larger DNA fragments
The Tools of Recombinant DNA Technology
• Vectors– Nucleic acid molecules that deliver a gene
into a cell
– Useful properties• Small enough to manipulate in a lab• Survive inside cells• Contain recognizable genetic marker• Ensure genetic expression of gene
– Include viral genomes, transposons, and plasmids
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Figure 8.3 Producing a recombinant vectorAntibioticresistancegene
Restrictionsite
mRNA for humangrowth hormone (HGH)
Reversetranscription
Plasmid (vector)
cDNA for HGH
Restrictionenzyme
Restrictionenzyme
Sticky ends
Gene for humangrowth hormone
Ligase
Recombinant plasmid
Introduce recombinantplasmid into bacteria.
Recombinantplasmid
Bacterialchromosome
Inoculate bacteriaon media containingantibiotic.
Bacteria containingthe plasmid withHGH gene survivebecause they alsohave resistance gene.
Step 3-Splice (or ligate) DNA into some kind of cloning vector to create a recombinant DNA molecule
Six different types of cloning vectors:
1. Plasmid cloning vector
2. Phage cloning vector
3. Cosmid cloning vector
4. Shuttle vectors
5. Yeast artificial chromosome (YAC)
6. Bacterial artificial chromosome (BAC)
7. Fosmid cloning vector
Plasmid Cloning Vectors:
Bacterial plasmids, naturally occurring small ‘satellite’ chromosome, circular double-stranded extrachromosomal DNA elements capable of replicating autonomously.
Plasmid vectors engineered from bacterial plasmids for use in cloning.
Feeatures (e.g., E. coli plasmid vectors):
1. Origin sequence (ori) required for replication.
2. Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only those cells with the anti-biotic resistance grow in colony).
3. Unique restriction site such that an enzyme cuts the plasmid DNA in only one place. A fragment of DNA cut with the same enzyme can then be inserted into the plasmid restriction site.
4. Simple marker that allows you to distinguish plasmids that contain inserts from those that do not (e.g., lacZ+ gene)
Some features of pUC19 plasmid vector:
1. High copy number in E. coli, ~100 copies/cell, provides high yield.
2. Selectable marker is ampR. Ampicillin in growth medium prevents growth of all other E. coli that do not contain plasmid.
3. Cluster of several different restriction sites called a polylinker occurs in the lacZ (-galactosidase) gene.
4. Cloned DNA disrupts reading frame and -galactosidase production.
5. Add X-gal to medium; turns blue in presence of -galactosidase.
6. Plaque growth: blue = no inserted DNA and white = inserted DNA.
7. Some % of digested vectors will reanneal with no insert. Remove 5’ phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques).
8. Plasmids are transformed into E. coli by chemical incubation or electroporation (electrical shock disrupts the cell membrane).
9. Good for <10kb; Cloned inserts >10 kb typically are unstable.
Phage cloning vectors:
1. Engineered version of bacteriophage (infects E. coli).
2. Central region of the chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted.
3. DNA is packaged in phage heads to form virus particles.
4. Phages with both ends of the chormosome and a 37-52 kb insert replicate by infecting E. coli.
5. Phages replicate using E. coli and the lytic cycle (see Fig. 3.13).
6. Produces large quantities of 37-52 kb cloned DNA.
7. Like plasmid vectors, large number of restriction sites available; phage cloning vectors useful for larger DNA fragments than pUC19 plasmid vectors.
Cosmid cloning vectors:
1. Features of both plasmid and phage cloning vectors.
2. Do not occur naturally; circular.
3. Origin (ori) sequence for E. coli.
4. Selectable marker, e.g. ampR.
5. Restriction sites.
6. Phage cos site permits packaging into phages and introduction to E. coli cells.
7. Useful for 37-52 kb.
Shuttle vectors:
1. Capable of replicating in two or more types of hosts..
2. Replicate autonomously, or integrate into the host genome and replicate when the host replicates.
3. Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells).
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Example:
*Insert firefly luciferase geneinto plasmid and transform Agrobacterium.
*Grow Agrobacterium in large quantities and infect tobacco plant.
Yeast Artificial Chromosomes (YACs):
Vectors that enable artificial chromosomes to be created and cloned into yeast.
Features:1. Yeast telomere at each end.
2. Yeast centromere sequence.
3. Selectable marker (amino acid dependence, etc.) on each arm.
4. Autonomously replicating sequence (ARS) for replication.
5. Restriction sites (for DNA ligation).
6. Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments.
Bacterial Artificial Chromosomes (BACs):
Vectors that enable artificial chromosomes to be created and cloned into E. coli.
Features:
1. Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors.
2. Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects.
3. Like other vectors, BACs contain:
1. Origin (ori) sequence derived from an E. coli plasmid called the F factor.
2. Multiple cloning sites (restriction sites).
3. Selectable markers (antibiotic resistance).
Fosmid:
1. Based on the E. coli bacterial F-plasmid.
2. Can insert 40 kb fragment of DNA.
3. Low copy number in the host (e.g., 1 fosmid).
4. Fosmids offer higher stability than comparable high copy number cosmids. Contain other features similar to plasmids/cosmids such as origin sequence and polylinker.
Creating Recombinant DNA Molecules
Cut DNA from donor and plasmid vector with the same restriction enzyme
Mix to generate recombinant DNA molecule
When such a modified plasmid is introduced into a bacterium, it is mass produced as the bacterium divides
Several techniques are used to insert DNA into cells- Chemicals that open transient holes in plasma membrane- Liposomes that carry DNA into cells
- Electroporation: A brief jolt of electricity to open membrane
- Microinjection: Uses microscopic needles - Particle bombardment: a gun like device
shoots metal particles coated with foreign DNA
Techniques of Recombinant DNA Technology
• Inserting DNA into Cells– Goal of DNA technology is insertion of DNA into
cell – Natural methods
• Transformation• Transduction• Conjugation
– Artificial methods• Electroporation• Protoplast fusion• Injection: gene gun and microinjection
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Figure 8.9a Artificial methods of inserting DNA into cells: electroporation
Chromosome
Electroporation
Pores in wall and membrane
Competent cell
Electricalfield applied
DNA fromanother source
Cell synthesizesnew wall
Recombinant cell
Figure 8.9b Artificial methods of inserting DNA into cells: protoplast fusion
Cell walls
Protoplast fusion
Polyethyleneglycol
Protoplasts
Enzymes removecell walls
Fused protoplasts
Recombinant cellNew wall
Cell synthesizesnew wall
Figure 8.9c Artificial methods of inserting DNA into cells: gene gun
Gene gun
Protoplasts
Nylonprojectile
Nylonprojectile
Blank .22caliber shell
DNA-coated beads
Vent
Target cell
Plate to stopnylon projectile
Figure 8.9d Artificial methods of inserting DNA into cells: microinjection
Microinjection
Target cell
Suction tubeto hold targetcell in place
Target cell’snucleus
Micropipettecontaining DNA
Selecting Recombinant Molecules
Three types of recipient cells can result from attempt to introduce a DNA molecule into a bacterial cell
1. Cells that lack plasmids
2. Cells with plasmids that do not contain foreign genes
3. Cells that contain plasmids with foreign genes
Selecting For Cells With Vectors
Vectors are commonly engineered to carry antibiotic resistance genes
Host bacteria without a plasmid die in the presence of the antibiotic
Bacteria harboring the vector survive
Growing cells on media with antibiotics ensures that all growing cells must carry the vector
Selecting For Cells With Inserted DNA
The site of insertion of the DNA of interest can be within a color-producing gene on the vector
Insertion of a DNA fragment will disrupt the vector gene
- And so the bacterial colony that grows will be colorless
Isolating Gene of Interest
Genomic library: Collections of recombinant DNA that contain pieces of the genome
DNA probe: Radioactively (or fluorescently) labeled gene fragments
cDNA library: Genomic library of protein encoding genes produced by extracting mRNA and using reverse transcriptase to make DNA
The Tools of Recombinant DNA Technology
• Gene Libraries– A collection of bacterial or phage clones
• Each clone in library often contains one gene of an organism’s genome
– Library may contain all genes of a single chromosome
– Library may contain set of cDNA complementary to mRNA
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Figure 8.4 Production of a gene library-overview
Genome
Isolate genomeor organism.
Generate fragments usingrestriction enzymes.
Insert each fragmentinto a vector.
Introduce vectorsinto cells.
Culture recombinant cells;descendants are clones.
Recombinant DNA Libraries (2 major types):
1. Genomic/chromosomal library, Collection of cloned restriction enzyme digested DNAs containing at least one copy of every DNA sequence in a genome or chromosome.
2. Complementary DNA (cDNA) library, Collection of clones of DNA copies made from mRNA isolated from cells.
reverse transcriptase (RNA dependent DNA polymerase) Synthesizes DNA from an RNA template cDNA libraries reflect what is being expressed in cells.
# of clones required for a complete library can be calculated from the size of the genome and average size of overlapping fragments cut by restriction enzymes.
Library should contain many times more clones than the calculated minimum number of clones.
Genomic Library:
3 ways to cut the DNA for a genomic library:
1. Complete digestion (at all relevant restriction sites)
1. Choice of restriction enzyme determines size of fragments (e.g., 4-base cutter gives smaller fragments, 8-base cutter gives larger fragments).
2. Produces a large number of non-overlapping DNA clones.3. Genes containing two or more restriction sites may be cloned in two or more
pieces.
2. Partial digestion
1. Limiting the amount and time the enzyme is active results in a population of overlapping fragments.
2. Fragments can be size selected by agarose electrophoresis.3. Fragments have sticky ends and can be cloned directly.
3. Mechanical shearing
1. Produces longer DNA fragments.2. Ends are not uniform, requires enzymatic modification before fragments can
be inserted into a cloning vector.
cDNA Library:
1. cDNA is derived from mature mRNA, does not include introns.
2. cDNA may contain less information than the coding region.
3. cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue and with time).
4. mRNA degrades quickly after cell death, and typically requires immediate isolation (cryoprotectants can increase yield if immediate freezing is postponed by fieldwork).
5. Creating a cDNA library:
1. Isolate mRNA
2. Synthesize cDNA
3. Clone cDNA
Applications of Recombinant DNA
Recombinant DNA is used to:- Study the biochemical properties or genetic pathways of that protein- Mass-produce proteins (e.g., insulin)
Sometimes conventional methods are still the better choice because of economics
Textile industry can produce indigo dye in E. coli by genetically modifying genes of the glucose pathway and introducing genes from another bacterial species
Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications
– Protein synthesis• Creation of synthetic peptides for cloning
– Vaccines• Production of safer vaccines• Subunit vaccines• Genes of pathogens introduced into common fruits and
vegetables• Injecting humans with plasmid carrying gene from
pathogen– Humans synthesize pathogen’s proteins
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Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications
– Genetic screening• DNA microarrays used to screen individuals for
inherited disease caused by mutations• Can also identify pathogen’s DNA in blood or
tissues
– DNA fingerprinting• Identifying individuals or organisms by their unique
DNA sequence
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Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications
– Gene therapy• Missing or defective genes replaced with normal
copies• Some patients’ immune systems react negatively
– Medical diagnosis• Patient specimens can be examined for presence of
gene sequences unique to certain pathogens
– Xenotransplants• Animal cells, tissues, or organs introduced into
human body© 2012 Pearson Education Inc.
Transgenic Animals
An even more efficient way to express some recombinant genes is in a body fluid of a transgenic animal
Transgenic sheep, cows, and goats have all expressed human genes in their milk, - Clotting factors- Clot busters- Collagen- Antibodies
Transgenic Animals
Finally, an organism must be regenerated from the altered cell
If the trait is dominant, the transgenic animal must express it in the appropriate tissue at the right time in development
If the trait is recessive, crosses between heterozygotes may be necessary to yield homozygotes that express the trait
Animal Models
Transgenic animals are far more useful as models of human diseases- Example: Inserting the mutant human beta globin gene that causes sickle-cell anemia into mice
Drug candidates can be tested on these animal models before testing on humans- Will be abandoned if they cause significant side effects
Animal Models
Transgenic animal models have limitations- Researchers cannot control where a transgene inserts, and how many copies do so- The level of gene expression necessary for a phenotype may differ in the model and humans- Animal models may not mimic the human condition exactly because of differences in development or symptoms
Bioremediation
Transgenic organisms can provide processes as well as products
Bioremediation: The use of bacteria or plants to detoxify environmental pollutants
Examples - Nickel-contaminated soils
- Mercury-tainted soils- Trinitrotoluene (TNT) in land mines
Applications of Recombinant DNA Technology
• Environmental Studies– Most microorganisms have never been grown
in a laboratory– Scientists know them only by their DNA
fingerprints• Allowed identification of over 500 species of
bacteria from human mouths• Determined that methane-producing archaea are a
problem in rice agriculture
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Applications of Recombinant DNA Technology
• Agricultural Applications– Production of transgenic organisms
• Recombinant plants and animals altered by addition of genes from other organisms
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Applications of Recombinant DNA Technology
• Agricultural Applications– Herbicide tolerance
• Gene from Salmonella conveys resistance to glyphosate (Roundup™)
– Farmers can kill weeds without killing crops
– Salt tolerance• Scientists have inserted gene for salt tolerance into
tomato and canola plants• Transgenic plants survive, produce fruit, and
remove salt from soil
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Applications of Recombinant DNA Technology
• Agricultural Applications– Freeze resistance
• Crops sprayed with genetically modified bacteria can tolerate mild freezes
– Pest resistance• Bt toxin
– Naturally occurring toxin harmful only to insects – Organic farmers used to reduce insect damage to
crops
• Gene for Bt toxin inserted into various crop plants• Genes for Phytophthora resistance inserted into
potato crops© 2012 Pearson Education Inc.
Applications of Recombinant DNA Technology
• Agricultural Applications– Improvements in nutritional value and yield
• Tomatoes allowed to ripen on vine and shelf life increased
– Gene for enzyme that breaks down pectin suppressed
• BGH allows cattle to gain weight more rapidly– Produce meat with lower fat content and produce
10% more milk
• Gene for β-carotene (vitamin A precursor) inserted into rice
• Scientists considering transplanting genes coding for entire metabolic pathways
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