Download Recombinant DNA Technology (Lecture 13)

Recombinant DNA Technology (Lecture 13)
Plant Biotechnology modification and use of the plants for improvement of human well being.
What plants can be good for?
1.Food
I.Improve taste
II.Improve the content of a particular part of the plant: oil content, fruits or roots size
III.Improve tolerance to biotic/abiotic stress
2.2.Construction material
3.Decoration
4.Medicaments
5.Vaccines
6.Biosensors
7.Phytoremediation
The technology used in the isolation or synthesis and joining together of unlike pieces of
DNA so that biologically active recombinant DNA molecules can be manufactured in the lab.
These recombinant DNA molecules can then be introduced into bacteria, yeasts, or other cells
where they can replicate and function (code for protein synthesis).
The basis of recombinant DNA technology is the ability to produce large amounts of a desired
piece of DNA. This is done by inserting the desired piece of DNA into a vector (cloning).
Microorganisms have already been programmed to produce a variety of medically useful
human proteins:
1. insulin (a hormone used to control diabetes),
2. erythropoietin (used to treat anemia by stimulating red blood cell production);
3. human growth hormone (somatotropin; used to treat growth deficiencies);
4. factor VIII (used to treat hemophilia);
5. alpha, beta, and gamma interferons (used against certain cancers and viral infections and as an
immune modulator; interferon-beta forslowing the progression of multiple sclerosis);
6. interleukin-2 (used as an immune enhancer and in adoptive immunotherapy);
7. granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor (promotes
the production of white blood cells; used to counter chemotherapy, improve resistance to infection, after
bone marrow transplants to treat leukemia; may be useful in treating infections by enhancing such
leukocyte functions as chemotaxis, diapedesis, phagocytosis, and extracellular killing);
8. tissue plasminogen activator (dissolves blood clots);
9. epidermal growth factor (help heal wounds, burns, and ulcers);
10. prourokinase (an anticoagulant used to treat heart attack);
11. monoclonal antibodies;
12. vaccine production (HBV, DaTP, HAV, Lyme disease, Rotavirus); and
13. bone morphogenic proteins (induce new bone formation following fractures and reconstructive
surgery).
Vectors
Plasmids:
•Small, autonomously replicating DNA molecules
•Plasmids are circular DNA molecules of 1-200 kb found in bacteria or yeast cells. They can be
considered molecular parasites but they often benefit their host (e.g. provide antibiotic
resistance)
•Plasmids used for cloning can replicate up to ~3,000 copies/cell _ they have a high copy number
•They contain conveniently located restriction endonuclease sites into which foreign DNA can be
inserted
•They are small and can be used to clone up to 10 kb
•They can replicate easily because they contain an origin of replication
•They carry genes specifying antibiotic resistance and _ can be selected
To obtain desired DNA
Three common methods include:
1. The fragmentation of total genomic DNA with restriction endonuclease enzymes. 2. Making a
DNA copy of cytoplasmic mRNA with the enzyme reverse transcriptase. 3. Synthesizing DNA
artificially in a machine if the exact base sequence is known.
DNA delivery
1.The desired DNA can be inserted into cloning vectors such as plasmids and viral genomes.
2.The desired DNA can be spliced into the genome of viruses capable of inserting their genome
into the chromosomes of the host cell, such as modified retroviruses or temperate
bacteriophages (gene transduction).
3. The desired DNA can be introduced into animal cells by microinjection and electroporation.
4. The desired DNA can be introduced into plant cells by protoplast fusion or using gene guns.
With protoplast fusion, the plant cell wall is enzymatically removed to create protoplasts.
Polyethylene glycol is then used to enable the protoplasts to fuse together. Gene guns use
helium bursts to propel microscopic gold or tungsten particles coated with DNA through plant cell
walls.
Bacteriophage
•Genome is double stranded linear DNA that gets packaged to form progeny phage
•Alternative cloning vehicle in which the central third of the 48 kb genome is not required for
infection and can be replaced by foreign DNA
Yeast artificial chromosomes (YACs)
•Linear DNA molecules that contain all the chromosomal structures required for normal
replication and segregation during yeast cell division
••Can clone up to several hundred kb fragments
Ligation
•The basis of cloning is the ability to join pieces of DNA together (ligation)
••First discovered in 1972 – a restriction fragment can be inserted into a cut made in a cloning
vector by the same restriction enzyme••DNA ligase covalently ligates or splices together the
sugar-phosphate backbone of the complementary ends of two DNAs
Transformation and Selection:
••Bacteria take up DNA (transformation) when DNA is mixed with treated bacteria and briefly
"heat shocked" at 42oC.
•Bacteria that contain the amp-resistant plasmid (~1 in 1000) will grow on agar plates containing
ampicillin, forming colonies
The Five Major Approaches to Molecular Biology:
A. Clone a new gene
B. Find out where it is expressed
C. Find a function for it
D. Analyze how the structure gives its function
E. Analyze how the promoter drives its expression
A. First step to cloning a new gene is making a library
A library is a set of fragments of DNA that represent the entire complement of DNA from the
organism of interest.
Genomic Libraries:
•Used for cloning entire genes (introns + exons)
•Useful for promoter analysis
cDNA Libraries:
•Used to clone cDNAs (no introns) for examination of the protein
Bacteriophage is used to make libraries instead of plasmids
•Packaging bacteriophage _and infecting bacteria is more efficient than transforming bacteria
with plasmid can screen more clones with bacteriophage
••Bacteriophage infection results in lysed bacteria which form "plaques" on a lawn of growing
bacteria plaques contain packaged bacteriophage DNA (containing one fragment of cDNA or
genomic DNA, depending on the library), viral protein and dead bacteria
••The packaged bacteriophage DNA or protein can bind to filters for subsequent analysis
There are many approaches to clone a new gene:
1. Clone by hybridization – need sequence information!
A. Look for genes that are homologous to a known protein. Make a radioactive probe from a
known cDNA and hybridize at low stringency to the library.
High stringency hybridization:
•Wash blot at high temperature (65oC) and low salt
•Only sequences that are >90% identical will remain hybridized under these conditions
Low stringency hybridization:
•Wash blot at lower temperature (55-60oC) and higher salt
•Sequences that are ~70-100% identical will remain hybridized
Have Xenopus (frog) MyoD cDNA and want to clone human MyoD – screen a human library with
the frog MyoD cDNA at low stringency.
E.g. Have human MyoD and want to look for family members – screen a human library with
human MyoD cDNA at low stringency
B. Look for the cDNA that corresponds to a purified protein. Get the amino acid sequence of the
purified protein and design degenerate oligonucleotides based on the protein sequence. Screen
the library at high stringency.
2. Clone by protein:
•For expression libraries, each plaque contains the protein encoded by the cDNA that is ligated
into the phage that formed the plaque.
•The library can be screened by any method that identifies a specific protein:
A.Antibodies against a specific protein can be used in a western blot protocol to identify plaques
containing the protein.
B.A DNA binding element from a promoter can be radiolabeled and used to screen the library for
proteins that bind it. This method can identify novel transcription factors.
C.Purified proteins can be radioactively labeled and used to screen a library for other proteins
that bind to it.
3. Clone by Differential Expression:
Fibroblasts have housekeeping genes but don’t have muscle-specific genes.
Skeletal muscle has housekeeping genes and muscle-specific genes (actin, myosin, troponin,
and muscle-specific transcription factors)
Subtractive cloning is a method used to isolate any clone present in one cell and not another.
Reverse Differential PCR analysis is the latest method for subtractive cloning.
B. Where is the clone expressed?
Northern blot analysis: Isolate RNA, separate by agarose gel electrophoresis, transfer to nylon,
probe with radioactive cDNA. Microchip Analysis:
Microchips contain 2,000 to 40,000 DNA sequences arranged in an array on a substrate.
The microchip is hybridized to fluorescently labeled cDNA obtained from a cell of interest.
The hybridized microchip is read in a reader and the genes expressed in the cell of interest are
identified
Applications of Microarray technology:
1. Gene discovery: The expression pattern of unknown genes may provide insight into
function.
2. Disease diagnosis: Ultimately, we will be able to obtain a readout of gene expression for
each patient. Treatment can be directly linked to different patterns of gene expression.
3. Drug discovery: Drug discovery can be linked to the genetic profiles of patients.
A)Recombinant Technology - genetic engineering - splicing genes from one organism into
another organism
B) Techniques
•viral transfer - using viruses to transfer genes
••restriction enzymes and plasmids in bacteria
••gene gun
••bacterial transformation and plants
C) Why splice genes from one organisms into another - Treat Disease
Drugs and hormones produced by recombinant bacteria
Using microbes to produce hormones or enzymes that individuals with genetic diseases can not
make for themselves
Splice normal human genes into bacteria which will make the desired product, involves using
restriction enzymes which cut the desired DNA sequence which is spliced on to bacteria
chromosomes (examples - Human growth hormone, insulin, interferon, Factor VIII, vaccines)
a)defective genes are replaced with normal genes, works for some genetic diseases, in the
experimental stages
b)b)Cystic Fibrosis - inhale viruses with the genes to correct salt problem in lung cells
c)c)SCID - WBC's are removed and cultured, infected with viruses containing normal genes,
reintroduced back into the body
1)Examples of transgenic plants with resistance to viruses
Tomato, potato and tobacco
2) Examples of transgenic plants with resistance to insects
cotton, corn
3) resistance to herbicides
4) retard spoilage in tomatoes
5) Supreme example - strawberry
resistance to drought, salt tolerance, insects, viruses, cold and frost, taste
DNA analyses and Genetic Fingerprinting
1)DNA fingerprinting in the courtroom
2)
Comparison of variable fragment lengths (RFLP's), cut by restriction enzymes, comparisons are
made by running electrophoretic gels
2)DNA and Wildlife Management
3)
Protection of Endangered Wildlife and Poaching
Project to describe DNA of all big game species for comparison with suspicious meat from poachers
or importers - genetic database
Theory: Complementarity and Hybridization
Molecular searches use one of several forms of complementarity to identify the macromolecules of
interest among a large number of other molecules.
Complementarity is the sequence-specific or shape-specific molecular recognition (binding of the two
strands of a DNA double-helix bind because of complimentary sequences) ;
Complementarity between a probe molecule and a target molecule can result in the formation of a probetarget complex.
This complex can then be located if the probe molecules are tagged with radioactivity or an enzyme.
The location of this complex can then be used to get information about the target molecule.
Hybrids of the following types can exist
1) DNA-DNA. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded, base-paired
hybrid with a ssDNA target if the probe sequence is the reverse complement of the target sequence.
2) DNA-RNA. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded, base-paired
hybrid with an RNA (RNA is usually a single-strand) target if the probe sequence is the reverse
complement of the target sequence.
3) Protein-Protein. An antibody probe molecule (antibodies are proteins) can form a complex with a
target protein molecule if the antibody's antigen-binding site can bind to an epitope (small antigenic
region) on the target protein. In this case, the hybrid is called an 'antigen-antibody complex' or 'complex'
for short.
Two important features of hybridization:
1)Hybridization reactions are specific - the probes will only bind to targets with complimentary
sequence (or, in the case of antibodies, sites with the correct 3-d shape).
2) Hybridization reactions will occur in the presence of large quantities of molecules similar but
not identical to the target. That is, a probe can find one molecule of target in a mixture of zillions of
related but non-complementary molecules.
Hybridization
These properties allow you to use hybridization to perform a molecular search for one DNA molecule, or
one RNA molecule, or one protein molecule in a complex mixture containing many similar molecules.
These techniques are necessary because a cell contains tens of thousands of genes, thousands of
different mRNA species, and thousands of different proteins.
Basic Definitions
Southern Blot
DNA cut with restriction enzymes - probed with radioactive DNA.
Northern Blot
RNA - probed with radioactive DNA or RNA.
Western Blot
Protein - probed with radioactive or enzymatically-tagged antibodies.
STEPS
•- Gel electrophoresis
••- Transfer to Solid Support
••- Blocking
••- Preparing the Probe
••- Hybridization
••- Washing
••- Detection of Probe-Target Hybrids
Gel Electrophoresis
•Staining DNA
DNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The DNA-EtBr complex
fluoresces under UV light.
•Staining RNA
RNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The RNA-EtBr complex
fluoresces under UV light.
•Staining Protein
Protein is stained with Coomassie Blue (CB). The protein-CB complex is deep blue and can be seen with
visible light.
Transfer to Solid Support
1) Electrophoresis, which takes advantage of the molecules' negative charge:
2) Capillary blotting, where the molecules are transferred in a flow of buffer from wet filter paper to dry
filter paper:
Blocking
•This coats the filter and prevents the probe from sticking to the filter itself.
••During hybridization, we want the probe to bind only to the target molecule.
Preparing the Probe
•1) The template DNA is denatured - the strands are separated - by boiling.
•2) A mixture of DNA hexamers (6 nucleotides of ssDNA) containing all possible sequences is
added to the denatured template and allowed to base-pair. They pair at many sites along each
strand of DNA.
•3) DNA polymerase is added along with dATP, dGTP, dTTP, and radioactive dCTP. Usually,
the phosphate bonded to the sugar (the a-phosphate, the one that is incorporated into the DNA
strand) is synthesized from phosphorus-32 (32P), which is radioactive.
•4) The mixture is boiled to separate the strands and is ready for hybridization.
Hybridization
•In all three blots, the labeled probe is added to the blocked filter in buffer and incubated for several hours
to allow the probe molecules to find their targets.
Washing
•To do this, the filter is rinsed repeatedly in several changes of buffer to wash off any un-hybridized
probe.
••The higher the wash temperature, the more stringent the wash, the fewer mismatches per hybrid are
allowed.
Detecting the Probe-Target Hybrids
•Autoradiography
•If the probe is radioactive, the radioactive particles that it emits can expose X-ray film. If you press the
filter up against X-ray film and leave it in the dark for a few minutes to a few weeks, the film will be
exposed wherever the probe bound to the filter. After development, there will be dark spots on the film
wherever the probe bound.
••Enzymatic Development
•If an antibody-enzyme conjugate was used as a probe, this can be detected by soaking the filter in a
solution of a substrate for the enzyme. Usually, the substrate produces an insoluble colored product (a
chromogenic substrate) when acted upon by the enzyme. This produces a deposit of colored product
wherever the probe bound.
Polymerase Chain Reaction
1.Separation of the two DNA chains in the double helix. ( 90-95C for 30’’).
2.But the primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 55 C.
At this temperature, the primers bind or "anneal" to the ends of the DNA strands. This takes about 20
seconds.
3.The final step of the reaction is to make a complete copy of the templates. Since the Taq polymerase
works best at around 75 degrees C (the temperature of the hot springs where the bacterium was
discovered), the temperature of the vial is raised.