BiotechnologyThe 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 90-95oCfor 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.

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.