Download - Biotechnology
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BiotechnologyThe New Frontier
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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
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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
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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.
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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.
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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.
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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
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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.
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Gene Cloning
• These new instructions are now passed along to the next generation of E. coli cells – This process
known as gene cloning.
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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
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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
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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.
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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.
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Gel Electrophoresis
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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
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Transfer via Electrophoresis
Electrophoresis takes advantage of the molecules' negative charge.
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Capillary Blotting
The molecules are transferred in a flow of buffer from wet filter paper to dry filter paper.
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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.
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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.
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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.
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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.
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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.
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Southern Blot
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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
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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
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Genomic Libraries
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Plasmid Biotechnology
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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
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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.
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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.
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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 90-95oCfor 30
seconds.
• Primers cannot bind DNA strands this high temperature– The vial is cooled to 55oC – 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 ~75oC – The temperature of the vial is raised.
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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.
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A Polymerase Chain Reaction
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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
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RFLP Allele Analysis
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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.
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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
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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
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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.
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• 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
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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.
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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.