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Biotechnology
 

Biotechnology

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    Biotechnology Biotechnology Presentation Transcript

    • Biotechnology The New Frontier
    • Recombinant DNA
      • Developed in the 1970’s at Stanford University
      • Researchers showed that genetic traits could be transferred from one organism to another
      • The DNA of one microorganism recombined with the inserted DNA sequence of another
      • The host DNA could thus be edited to exhibit a specific modification
      • This process is similar to editing a written text:
        • scissors and "glue" are used to "cut" and "paste."
      • This process can be used to produce many protein products of medical and economic importance
    • The Insulin Example
      • The gene for insulin production in humans could be cut and then pasted into the DNA of E. coli, a bacterium that inhabits the human digestive tract.
      • Bacterial cells divide rapidly making billions of copies
      • Bacteria can be raised in a laboratory in tissue culture media.
      • Each bacterium carries in its DNA a replica of the gene for insulin production.
        • Each new E. coli cell has inherited the human insulin gene “sentence.”
      • Human insulin is produced by the bacteria and can be harvested from the growth medium
    • Transferring Genes
      • How do we transfer the gene carrying the instructions for insulin production?
      • One method is to cut the gene from human DNA and paste, or splice, it into plasmid DNA
        • Plasmids are a special type of bacterial DNA that takes a circular form and can be used as a vehicle for this editing job.
      • Our "scissors" are the class of enzymes called restriction enzymes .
    • Restriction Enzymes
      • Also called restriction endonucleases
      • There are over a hundred restriction enzymes
      • Each enzymes cuts in a very precise way at a specific base sequence of the DNA molecule.
      • With these “scissors” used singly or in various combinations, the segment of the human DNA molecule that specifies insulin production can be isolated.
    •  
    • Production of Recombinant DNA
      • This excised segment of DNA is "glued" into place using the enzyme DNA ligase.
      • The result is an edited, or recombinant , DNA molecule.
    •  
    •  
    • Cloning a Human Gene
      • This is a highly simplified description of rDNA technology
      • A recombinant bacterial plasmid can be created carrying the gene of interest
    • Forming a Recombinant Plasmid
      • When this recombinant plasmid DNA is inserted into E. coli, the cell will be able to process the instructions to assemble the amino acids for insulin production.
    • Gene Cloning
      • These new instructions are now passed along to the next generation of E. coli cells
        • This process known as gene cloning .
    • Molecular “Searching” Techniques
      • Techniques for analyzing DNA, RNA, and protein.
      • Molecular searches use one of several forms of complimentarity to identify target macromolecules among a large number of other molecules.
      • Major Techniques include:
      • Southerns blots,
      • Northern blots
      • Western blots
      • Cloning
    • Techniques
      • 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.
    • Complimentarity and Hybridization
      • Complementarity is the sequence-specific or shape-specific molecular recognition that occurs when two molecules bind together.
        • The two strands of a DNA double-helix bind because they have complimentary sequences
        • An antibody binds to a region of a protein molecule (antigen) because they have complimentary shapes .
      • Complementarity between a probe molecule and a target molecule can result in the formation of a probe-target complex.
    • The Probe-Target Complex
      • The probe-target complex can then be located if the probe molecules are tagged with radioactivity or an enzyme.
      • The location of the complex can be used to get information about the target molecule.
      • The probe target complex, formed from two types of molecules, is called a hybrid
      • In solution, several types of hybrid molecular complexes (hybrids) can exist
    • Hybrid Types
      • DNA-DNA
        • A single-stranded DNA (ssDNA) probe can form a double-stranded, base-paired hybrid with a ssDNA target if the probe sequence is complimentary to the target sequence.
      • DNA-RNA.
        • A single-stranded DNA (ssDNA) probe can form a double-stranded, base-paired hybrid with an RNA target if the probe sequence is complimentary to the target sequence.
      • Protein-Protein.
        • An antibody (Ab) probe can form a complex with a target protein if the antibody's antigen-binding site can bind to an epitope (small antigenic region) on the target protein.
        • This type of hybrid is called an 'antigen-antibody complex' or 'complex' for short.
    • Features of Hybridization
      • Hybridization reactions are specific
        • probes will only bind to targets with complimentary sequence (or, in the case of antibodies, sites with the correct 3-d shape).
      • Hybridization reactions will occur in the presence of large quantities of molecules similar but not identical to the target.
        • A probe can find one molecule of target in a mixture of zillions of related but non-complementary molecules.
      • These properties allow 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.
    • The Importance of Hybridization
      • Hybridization techniques allow you to pick out the molecule of interest from the complex mixture of cellular components and study it on its own.
      • These techniques are necessary because a cell contains tens of thousands of genes, thousands of different mRNA species, and thousands of different proteins.
      • When the cell is broken open to extract DNA, RNA, or protein, the result is a complex mixture of all the cell's DNA, RNA, or protein.
    • Locating the Target
      • If you mix a solution of DNA with a solution of radioactive probe, you end up with a radioactive solution.
        • You cannot tell the hybrids from the non-hybridized molecules.
      • First you must physically separate the mixture of molecules on the basis of some convenient parameter.
      • The molecules must then be immobilized on a solid support, so that they will remain in position during probing and washing.
      • The probe is then added, the non-specifically bound probe is removed, and the probe is detected.
      • The place where the probe is detected corresponds to the location of the immobilized target molecule.
    • Steps in the Process
      • The process has the following steps:
      • Gel electrophoresis
      • Transfer to solid support
      • Blocking
      • Preparing the probe
      • Hybridization
      • Washing
      • Detection of probe-target hybrids
    • Separation by Molecular Weight
      • In Southern, Northern, and Western blots, the initial separation of molecules is on the basis of molecular weight.
      • Cloning uses a different technique
      • Gel electrophoresis is the usual procedure used
      • This separates molecules on the basis of their size.
    • Gel Electrophoresis
      • First, a slab of gel material is cast.
        • Gels are usually cast from agarose or poly-acrylamide.
        • Gels are solid and consist of a matrix of long thin molecules forming sub-microscopic pores.
        • The size of the pores can be controlled by varying the chemical composition of the gel.
      • The gel is placed in a tank holding buffer and equipped with electrodes to apply an electric field
      • The properties of the buffer & pH are set so that the molecules being separated carry a net (-) charge
      • The charged molecules are moved by the electric field from left to right.
    • Gel Electrophoresis
    • Separation of Molecules on a Gel
      • As they move through the gel, larger molecules will be restricted the pores of the gel, while the smaller molecules will move more easily and thus faster.
      • This results in a separation by size, with the larger molecules nearer the well and the smaller molecules farther away.
      • This separates on the basis of size, not necessarily molecular weight.
    • Size vs. Molecular Weight
      • Example: two 1000 nucleotide RNA molecules, one a fully extended long chain ( A ); the other can base-pair with itself to form a hairpin structure ( B ):
      • As they migrate through the gel, both molecules behave as though they were solid spheres whose diameter is the same as the length of the rod-like molecule.
      • Both have the same molecular weight
      • B has secondary structure making it smaller than A
      • B will migrate faster than A in a gel
    • Controlling for Shape
      • To prevent differences in shape from confusing measurements of molecular weight, the molecules to be separated must be in a long extend rod conformation
      • Different techniques are used to remove secondary or tertiary structure, in preparing DNA, RNA and protein samples for electrophoresis.
    • Preparation Techniques
      • Preparing DNA for Southern Blots
        • DNA is first cut with restriction enzymes
        • The resulting double-stranded DNA fragments have an extended rod conformation without pre-treatment.
      • Preparing RNA for Northern Blots
        • Although RNA is single-stranded, RNA molecules often have small regions that can form base-paired secondary structures.
        • RNA is pre-treated with formaldehyde.
      • Preparing Proteins for Western Blots
        • Proteins have extensive 2' and 3' structures and are not always negatively charged.
        • Proteins are treated with detergent (SDS) which removes 2' and 3' structure and coats the protein with negative charges .
    • Determining Molecular Weight
      • Molecules will be separated by molecular weight
      • The distance migrated is ~proportional to the log of the inverse of the molecular weight (the log of 1/MW).
      • Molecular weights are measured in different units for DNA, RNA, and protein:
      • DNA
        • Molecular weight is measured in base-pairs (bp) and commonly in kilobase-pairs (1000bp), or kbp.
      • RNA
        • Molecular weight is measured in nucleotides (nt) and commonly in kilonucleotides (1000nt), or knt.
      • Protein
        • Molecular weight is measured in Daltons (grams per mole)(Da), and commonly in kiloDaltons (1000Da), or kDa.
    • Use of Standards
      • On most gels, one well is loaded with a mixture of DNA, RNA, or protein molecules of known molecular weight.
      • These 'molecular weight standards' are used to calibrate the gel run
      • The molecular weight of any sample molecule can be determined by interpolating between the standards.
    • Staining
      • Different stains and staining procedures are used for different classes of macromolecules:
      • DNA & RNA
        • DNA and RNA are stained with ethidium bromide (EtBr), which binds to nucleic acids.
        • The nucleic acid-EtBr complex fluoresces under UV light.
      • 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
      • After the DNA, RNA, or protein has been separated by molecular weight, it must be transferred to a solid support before hybridization.
        • Hybridization does not work well in a gel.
      • The transfer process is called blotting
        • These hybridization techniques are called blots.
      • The solid support is often a sheet of nitrocellulose paper (a type of filter paper)
        • DNA, RNA, and protein stick well to nitrocellulose in a sequence-independent manner.
      • The DNA, RNA, or protein can be transferred to nitrocellulose in one of two ways:
        • Electrophoresis
        • Capillary blotting
    • Transfer via Electrophoresis Electrophoresis takes advantage of the molecules' negative charge .
    • Capillary Blotting The molecules are transferred in a flow of buffer from wet filter paper to dry filter paper.
    • Blocking
      • The surface of the filter now has the separated molecules on it, as well as many spaces where no molecules have yet bound.
      • If we added the probe directly to the filter, the probe would stick to the blank parts of the filter, like the molecules transferred from the gel did.
      • During hybridization, we want the probe to bind only to the target molecule.
      • To achieve this, the filters are soaked in a blocking solution which contains a high concentration of DNA, RNA, or protein.
      • This coats the filter and prevents the probe from sticking to the filter itself.
    • Preparing the Probe
      • Radioactive DNA probes for Southerns and Northerns
        • A radioactive copy of a double-stranded DNA fragment.
        • Begins with a restriction fragment of a plasmid containing the gene of interest.
        • The DNA restriction fragment (template) is radiolabeled
        • This produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe.
    • Probes for Westerns
      • Radioactive Antibodies for Westerns
        • Antibodies are raised by injecting a purified protein into an animal, usually a rabbit or a mouse.
        • This produces an immune response to that protein.
        • Antibodies isolated from the serum (blood) of that rabbit will bind to the protein used for immunization.
        • Antibodies are protein molecules
        • They are labeled by chemically with iodine-125 which is radioactive.
      • Enzyme-conjugated Antibodies for Westerns
        • Antibodies against a particular protein are raised as above
        • These are labeled by chemically cross-linking the antibody molecules to molecules of an enzyme.
        • The resulting antibody-enzyme conjugate is still able to bind to the target protein.
    • Hybridization & Washing
      • In all three blots, the labeled probe is added to the blocked filter paper in buffer to allow hybridization
      • This is incubated for several hours to allow the probe molecules to find their targets.
      • After hybrids have formed it is necessary to remove any probe that is on the filter but not stuck to the target molecules.
      • To do this, the filter is rinsed repeatedly in several changes of buffer to wash off any un-hybridized probe.
    • Detecting the Probe-Target Hybrids
      • The nitrocellulose looks like blank paper
        • You must now detect where the probe has bound.
      • Autoradiography
        • If the probe is radioactive, it can expose X-ray film.
        • X-ray film is pressed against 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.
        • The substrates used produce colored product when acted on by the enzyme.
        • This produces a colored deposit wherever the probe bound .
    • Southern Blot
    • Cloning a Gene by Hybridization :
      • We want to end up with a plasmid which contains a fragment of human DNA which includes the gene of interest
      • We can not isolate the human gene DNA from either the gel or the filter
        • At each molecular weight on the gel, there are many bands of the same length but different sequences.
        • Therefore, separating the DNA fragments by molecular weight is unsuitable.
      • Instead, we separate them by sequence
    • Constructing a Genomic Library
      • We create a collection of plasmids, physically separated, each containing a different fragment of human DNA.
      • This is a plasmid library of human DNA restriction fragments.
      • The plasmids are transferred to bacteria
      • This results in a collection of bacterial colonies, each containing a different plasmid with a different inserted piece of human DNA
      • These bacteria are grown on agar plates
      • Previously described searching techniques can then be used to isolate the gene of interest
    • Genomic Libraries
    • Plasmid Biotechnology
    • Polymerase Chain Reaction
      • The polymerase chain reaction (PCR) is widely used in research laboratories and doctor's offices
      • This techniques “xeroxes” DNA
      • PCR mimics the process of DNA replication in a test tube.
      • PCR relies on the ability of DNA-copying enzymes to remain stable at high temperatures.
      • PCR uses polymerase derived from Thermus aquaticus, a bacterium found in hot springs in Yellowstone Park
        • Extremely heat tolerant
    • Polymerase Action
      • When a cell divides, polymerase enzymes make a copy of the DNA in each chromosome.
      • The first step in this process is to "unzip" the two DNA chains of the double helix.
      • As the two strands separate, DNA polymerase makes a copy using each strand as a template.
    • Elements of a PCR
      • PCR entails 3 steps:
        • Separation of the strands
        • Annealing the primer to the template
        • Synthesis of new strands
      • All three steps are carried out in the same vial.
      • The entire process takes less than two minutes.
      • A PCR vial contains all the necessary components for DNA duplication:
        • A piece of DNA
        • Large quantities of the four nucleotide
        • Large quantities of the primer sequence
        • DNA polymerase.
    • Temperature Dependent Steps
      • The 3 parts of the PCR are carried out in the same vial, but at different temperatures.
      • The first part of the process separates the two DNA chains in the double helix.
        • This is done by heating the vial to 90-95 o Cfor 30 seconds.
      • Primers cannot bind DNA strands this high temperature
        • The vial is cooled to 55 o C
        • At this temperature, the primers bind or "anneal" to the ends of the DNA strands.
      • The final step is to make a complete copy of the templates.
        • The Taq polymerase works best at ~75 o C
        • The temperature of the vial is raised.
    • The PCR Process
      • Taq polymerase begins adding nucleotides to the primer
      • This continues until a complete, complimentary strand is produced
      • At the end of a cycle, each piece of DNA in the vial has been duplicated.
      • Each newly synthesized DNA piece can act as a new template
        • The cycle can be repeated 30 or more times.
        • After 30 cycles, 1 million copies of a single piece of DNA can be produced!
        • 1 million copies can be ready in about three hours.
      • Small samples of DNA can produce sufficient copies to carry out forensic tests.
    • A Polymerase Chain Reaction
    • DNA Fingerprinting
      • Each person has a unique DNA fingerprint.
      • A DNA fingerprint is the same for every cell, tissue, and organ of a person.
      • It cannot be altered by any known treatment.
      • DNA fingerprinting is a quick way to compare the DNA sequences of any two living organisms.
      • DNA fingerprinting has become the primary method for identifying and distinguishing among individual human beings.
      • An additional application of DNA fingerprint technology is the diagnosis of inherited disorders
    • RFLP Allele Analysis
    • Steps in DNA Fingerprinting
      • 1. Isolation of DNA
        • DNA is recovered from cells or tissues of the body.
        • Only a small amount of tissue is needed.
        • The amount of DNA found at the root of one hair is usually sufficient.
      • 2. Cutting, sizing, and sorting
        • Restriction enzymes are used to cut the DNA at specific places.
        • The DNA pieces are sorted according to size by gel electrophoresis.
    • Steps in DNA Fingerprinting, Cont .
      • 3. Transfer of DNA to nylon
        • The distribution of DNA pieces is transferred to a nylon sheet by placing the sheet on the gel and soaking them overnight.
      • 4-5. Probing
        • Adding radioactive or colored probes to the nylon sheet produces a pattern called a DNA fingerprint.
        • Each probe typically sticks in only one or two specific places on the nylon sheet.
      • 6. DNA fingerprint
        • The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously.
        • It resembles the bar codes used by grocery store scanners
    • Uses of DNA Fingerprints
      • DNA fingerprints are useful in several applications of human health care research, as well as in the justice system.
        • Used to establish paternity
        • Used to identify criminal suspects
        • Used as a diagnostic tool for inherited disorders
    •  
    • Diagnosis of Inherited Disorders
      • DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies
        • These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others.
      • Early detection enables the medical staff to prepare themselves and the parents for proper treatment of the child.
      • Genetic counselors can use DNA fingerprint information to help prospective parents understand the risk of having an affected child.
        • Prospective parents may use DNA fingerprint information in their decisions concerning affected pregnancies.
      • Even if the precise locus of a disease causing allele is unknown, its presence can be often be detected
      • Test for RFLP markers that are close to the gene of interest
      • Basis of sometimes family-specific genetic testing
    • Developing Cures for Inherited Disorders
      • Research to locate inherited disorders on specific chromosomes depends on the information contained in DNA fingerprints.
      • By studying the DNA fingerprints of relatives who have a history of a particular disorder, or by comparing large groups of people with and without the disorder, it is possible to identify DNA patterns associated with the disease
      • This work is a necessary first step in designing an eventual genetic cure for these disorders .
    • Personal Identification
      • Every organ or tissue of an individual contains the same DNA fingerprint
      • The U.S. armed services has begun a program to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action.
      • The DNA method will be far superior to the dogtags, dental records, and blood typing strategies currently in use.