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
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 .
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.
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:
DNA cut with restriction enzymes
probed with radioactive DNA
probed with radioactive DNA or RNA.
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
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.
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.
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:
Transfer to solid support
Preparing the probe
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.
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.
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.
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:
Molecular weight is measured in base-pairs (bp) and commonly in kilobase-pairs (1000bp), or kbp.
Molecular weight is measured in nucleotides (nt) and commonly in kilonucleotides (1000nt), or knt.
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.
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 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:
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.
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.
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.
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 .
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
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
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
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
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.
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 .
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.