Download Recombinant DNA

Chapter 13
Genetic Engineering
and
Recombinant DNA
Key Questions
• What does it mean to “genetically engineer” an
organism?
• What steps must biologists take to insert a gene
into the genome of another organism?
• How are transgenic plants and animals useful
to biologists and others?
Discovery of PCR
• Kary Mullis: inventor of PCR machine
– Not in the mold of a usual Nobel Prize winner.
– Manages to offend nearly everyone.
– Hasn’t worked in a lab for years.
– Went to UC Berkeley to get a Ph.D. in biochemistry. Enjoyed the climate
at Berkeley.
– Several failed marriages, little success in finding a job.
– Eventually worked as a lab technician at Cetus Corporation.
• Invention of PCR:
– While thinking of ways to make short pieces of DNA easier, he thought of
a new technique which came to be called polymerase chain reaction or
“PCR.”
– In theory he should be able to take out one specific string of nucleotides
from a DNA strand and make millions of copies of that one specific
sequence.
Discovery of PCR
• Invention of PCR:
– Make an oligonucleotide and bind it to a long strand of DNA.
– Oligonucleotide serves as primer for DNA polymerase.
– Polymerase would make a copy of the down-stream sequence of DNA.
– Then use the newly-made strand to make another strand.
– Then a chain reaction – number of copies would go up rapidly – 2, 4, 8,
16, etc.
• Mullis worked the method out with other workers at Cetus.
• Very valuable for finding a particular sequence and making many thousands
of copies for molecular biologists to work with
• Mullis and PCR:
– Mullis could not get along with other Cetus employees.
– He thought he deserved more credit.
– Now he does little or no science.
– But his invention contributed to many research careers.
– Nobel Prize in 1993.
PCR
• Selectively copying a short segment of DNA
PCR Depends on Primers
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Biotechnology
Biotechnology: use of scientific and engineering principles to manipulate
organisms to
• achieve specific characteristics:
biochemical, morphological or growth
• produce useful products
Beer, vinegar, yogurt, cheese
• obtain information about an organism or tissue
Mode of action, future engineering
Biotechnology selects useful traits from a range of variation
– Traditional examples: domestic animal breeding, plant breeding, beer
brewing, cheese making
– Today increased precision and speed, but only those traits that are
biochemically understood, not complex traits
Natural and Induced Variation
Example of penicillin.
– Effective antibiotic but in low concentrations in the mold that makes it.
– Searched soil samples to find the most productive penicillin-forming
mold.
– Induced mutations in the strains with high penicillin.
– Combining natural and induced variability, selected a strain with notably
higher penicillin production, eventually increase production 100X
Penicillin Resistance and
Sensitivity
Classical Biotechnology
Humans have manipulated other organisms for 10,000 years to select and
maintain characteristics of interest and importance
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Bacteria, yeasts, other fungi: used to make cheese, yogurt, alcoholic
beverages, sauerkraut, bread, even fight infections
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Advances in microbiology in 19th Century : fermentation, pure culture
methods, microscopy, understanding of microbial pathogenesis, vaccination
etc. led to antibiotics and vaccines (smallpox天花, cholera霍亂, diphtheria白
喉, tetanus破傷風)
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Modern uses : antibiotic production, modern industrial processes (food
industry, pharmaceutical industry)
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Based on understanding of variability among organisms, and ability to
select the most valued variant for a given function: disease resistance or
microorganisms useful in fermentation of substrates or manufacture of
industrial products
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Animals and plants subjected to selective breeding to produce “better”
strains (接枝育種): plants with hybrid vigor, higher yield
Growing Virus in Eggs
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Recombinant
DNA
Recombinant DNA: Any DNA molecule composed of DNA from two or more
sources not found together in nature
– The first recombinant DNA:SV40 from monkey virus is joined w/ λ phage
DNA to form the first recombinant DNA by Paul Berg in 1972
• Applications of Genetic Engineering
– The production of proteins for therapeutic use
– The development of genetically engineered vaccines
– Gene therapy
– Food biotechnology
– Ethical guidelines for genetic engineering
The Production of Proteins for
Therapeutic Use
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Molecular techniques produce medically important human proteins in
bacteria
Previously difficult to isolate directly from human sources
First proteins in 1970’s
· human insulin to treat diabetes
· human growth hormone to treat pituitary dwarfism
Cloning and Expression of a gene
• Find the gene of interest (genomic or cDNA cloning)
• Cut DNA into gene fragments using restriction endonuclease
• Cut vector (plasmid) with same enzyme
Recombinant DNA
• Restriction Enzymes
– Cut DNA only at specific sequences.
– Specific sequence called a restriction site.
– A restriction map can be made of all the places cut with a certain
restriction enzyme.
– Some cut the DNA in a staggered way so that the DNA has sticky ends.
– Rejoin ends with DNA ligase.
• Joining DNA of two species – a bacterium and a human, for example.
– Need a plasmid that will enter a bacterial cell.
– First the DNA from the plasmid and the human is cut with the same
restriction enzyme.
– Make a combination of human gene and a plasmid with no more than a
replication site and a gene for antibiotic resistance.
– Plasmid part of recombinant DNA carries human part into bacterial cells.
Recombinant DNA
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Several kinds of vectors in addition to bacterial plasmids expand the range
of combinations of DNA.
• Bacteriophages.
• Animal and plant viruses.
• Transposable elements.
• Fragments of chromosomes.
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Getting bacteria to express eukaryotic genes.
• Bacteria lack introns.
• Lack ability to remove introns from RNA transcribed by eukaryotic
genes.
• Use retroviruses – they have reverse transcriptase.
• Copy a messenger RNA to make an intron-less DNA called cDNA.
• Then put that DNA into a bacterial host.
• Expression of a eukaryotic gene.
Recombinant DNA
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Cut and paste DNA fragments
Or replace single nucleotides
Use viruses or plasmids to move into new cells
Make copies in bacteria or other cells
Restriction Enzymes
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Cut DNA only at specific sequences
Sequence is a restriction site
Restriction fragments are DNA pieces that all have same end sequences
Restriction map is the sites on a DNA molecule cut by a particular restriction
enzyme
Restriction Enzyme
DNA Agarose Gel Electrophoresis
Restriction Digestion and
Ligation of DNA
Sticky Ends
• Staggered cuts leave sticky ends that can be joined by ligase
Cloning Human Insulin
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Cloning genes: Splice insert and plasmid DNA together; uptake into recipient
cell by transformation
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Screening clone: use probe to detect specific gene or protein sequence to
identify clone
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Sequencing the clone: for gene coding region identification
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PCR amplification of gene fragment: for use in research applications and
analysis, medical diagnosis, military applications, forensic medicine
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Expressing the gene: for protein production
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Protein purification: for analysis and therapeutic application
Molecular Cloning of Human Insulin
Molecular Cloning of Human Insulin
Eukaryotic Genes in Bacteria
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Eukaryotic genes have introns, but bacteria cannot process mRNA
Use processed mRNA for cloning
RNA copied into DNA with reverse transcriptase enzyme
Resulting DNA is a copy of mRNA, or cDNA, without introns
Join to vector; put into a cell
Multiplying Recombinant DNA
Recombinant plasmids are copied as host cells replicate
PCR used to make millions of copies of a specific sequence, without
constructing recombinant DNA molecule
PCR requires knowledge of the sequence
• Make short polynucleotides that correspond to the sequences
of the ends of the gene of interest.
• PCR machine heats DNA, causing the strands to separate.
• Cool the mixture, each single strand directs the synthesis of
another.
• Heat mixture again, separate. Then cool again, synthesize.
Continue this until lots of new DNA has been made.
Eukaryotic Genes in Bacteria
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Expressing eukaryotic genes.
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Often bacteria will not express introduced genes from eukaryotes.
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Bacteria lack the appropriate regulatory mechanisms.
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So vectors are used that will put genes in other eukaryotic cells.
• Many products have been made.
Some proteins made by recombinant DNA are so low in concentration in
nature that they are being studied now for the first time.
Modified Proteins
– Some eukaryotic proteins are modified after translation, bacteria
cannot do modifications
– Insert new genes into eukaryotic cells:
• Moth, baculovirus system
• Yeast
• Mammalian cells in culture
Recombinant DNA Products
• Human insulin, growth hormone
• Tissue plasminogen activator
• Erythropietin
• Vaccines
• Cytokines
Gene Libraries
• Gene Library: all the pieces of DNA after a genome has been treated with
restriction enzymes
– Thousands or millions of restriction fragments from a single genome
– Each fragment combined with a vector
– Recombinant molecule introduced into bacteria
– Library contains at least 1 copy of every sequence in the genome
– Molecular hybridization: Use a piece of DNA from a known sequence as
a probe to find the complementary sequence in the library.
– Use antibodies to a known protein as a probe to find the sequence in the
library that expresses that particular gene.
• cDNA Library
– Constructed from mRNA
– Contains only expressed genes from a specific cell type or tissue
• Finding a Gene in a Library
– Use a probe, a short single strand of DNA complementary to part of the
gene wanted
– Or, if the protein product is known, use an antibody that recognizes the
protein
– Find 1 colony in 500,000 with the gene of interest
Locating a Gene from a DNA Library
Southern Blotting
• Used to look at a small number of fragments in a complex mixture
– Separate restriction fragments by electrophoresis
– Immobilize fragments on filter paper
– Hybridize filter with radioactive probe
– Expose to X-ray film
Types of Blots
• Southern: visualize
DNA fragments
• Northern: visualize RNA
molecules to determine
gene expression in a
cell type
• Western: visualize
proteins using
antibodies as probes
Conventional DNA Sequencing
Modern DNA Sequencing
Transgenic Organisms
• Transgenic organisms: Organisms that carry recombinant DNA in their
genomes
• New DNA inserted into fertilized egg — during development all cells in the
body will have new gene
• Inefficient process — 1% success
• Requires normal expression and must not disrupt other genes
• First Transgenic Animals
• Mice were given human growth hormone, grew twice as big
• Later — pigs, goats, sheep and monkeys
Process of Making Transgenic Animals
Transgenic and Cloned Animal
Knockout Animals
• Experimental animals with
inactivated gene
– Used to study genetic
diseases
– Determine function of a
gene
• Many surprises
– Long haired mice
• Or no effect
Genetically Engineered Plants
• Easier to modify plants than
animals
– Ti plasmid vector carries
genes into plants
– Gold pellets coated with
DNA shot into cells with a
gene gun
• Cells grown in culture can
become viable plants
• Bt corn contains bacterial
toxin — kills insects
• Fungus resistant soy beans
• Round-up Ready plants
resistant to herbicide Roundup
Genetic Engineering of Plants
Genetic Engineering of Plants
Genetic Engineering of Plants
Genetic Engineering of Plants
DNA Microarrays
• Used to study expression of thousands of genes at 1 time
• Thousands of DNA fragments arranged on a filter or chip
Using Microarrays
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Isolate mRNA from cells, copy into cDNA with fluorescent tag
Hybridize cDNA with microarray
Expressed genes light up spots on the chip
Can be quantitated — bright spots, high expression levels in cell
Compare patterns of gene expression in cancer and normal cells — which
genes are turned on in cancer cells?
• Cluster analysis — find groups of genes that are regulated together
• Analysis is called bioinformatics
DNA Microarray
cDNA
or
Oligo DNA
FITC, Cy3
Cy5
Glass, silicon, nylon membrane
The Proteomic Era
The Proteomic Era
1183.6498
1199.6755
1457.6913
1552.7802
1045.5584
1000
1894.9346
1909.9414
1488.7815
1876.9218
1485.7244
1675.7370
1430.7831
1500
Mass (m/z)
2000
2299.1675
2167.1922
2599.2658
2500
Extract peptides;
MALDI-TOF analyze
Run 2D gel;
Stain/Image
Edman Degradation
AAA Composition
Immunoblot
MS
Excise spot;
wash; digest
Database search
Bioengineered Products
• Bioengineered products:
– High-oil corn
– Soy beans with increased lysine and methionine (low levels in grain)
– Sunflowers with more oil
– Tobacco plants make human antibodies
– Tomatoes make serum albumin for burn victims
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Risks of Genetically Modified (GM) Plants
– Gene exchange with wild plants (herbicide resistance), either through
conventional interbreeding or viruses or mobile genes
– Dispersing bacterial or viral genes that cause plant diseases
– Dispersing genes for antibiotic resistance in the food supply
– Using gene promoter that can function in all plants, green algae, yeasts,
and bacteria
Example of Escaping Gene
• Rapeseed (source of canola oil) resistant to herbicide fed to bee
larvae
• Bacteria and yeast from gut of bee carried genes for herbicide
resistance
• Engineered genes can move between organisms
• Economic Issues
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GM plants are patented
Farmers must buy new seed each year
Cannot save seed from 1 crop to plant the next year
Farmers in developing countries become economically dependent