Introduction

Biotechnology refers to technology used to manipulate DNA. The procedures are often referred to as genetic engineering.

DNA is the genetic material of all living organisms and all organisms use the same genetic code. Genes from one kind of organism can be transcribed and translated when put into another kind of organism. 

For example, human and other genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use. Human insulin, human growth hormone, and vaccines are produced by bacteria.

Recombinant DNA refers to DNA from two different sources.  Individuals that receive genes from other species are transgenic.

Biotechnology techniques often involve putting genes in viruses or bacteria.

Viruses contain genetic material but are not living. Host cells are required for their reproduction.

Viruses are composed of an inner nucleic acid core (genetic material) and an outer protein coat (capsid).

An outer envelope (membrane) that is derived from membranes of the host cell may surround the capsid.

The genetic material in some viruses is DNA; in others it is RNA.

When viral genetic material enters a cell, it is replicated, transcribed (mRNA is produced) and translated (proteins are produced from the mRNA) by the host cell. By this process, the host cell uses the genetic instructions in the virus to make more viruses.

viral DNA ® mRNA ® protein

If the viral genetic material is RNA, a DNA copy must first be made before transcription and translation can occur. The DNA copy of the viral RNA is called cDNA.

Retroviruses

The genetic material of retroviruses is RNA. The retrovirus carries an enzyme called reverse transcriptase, which is capable of creating a DNA copy of the viral RNA. 

The new DNA produced from the RNA template is called cDNA.

viral RNA ® cDNA ® mRNA ® protein

DNA synthesis follows the production of cDNA to produce a double-helix.

The AIDS virus (HIV) is an example of a retrovirus.

Recombinant DNA contains DNA from 2 or more different sources.

Vectors are pieces of DNA that are used to transfer genes into a host cell.

Marker genes can be used to determine if the gene has been taken up. Marker genes must have some distinguishable characteristic. For example if you put a gene that enables bacteria to be resistant to the antibiotic ampicillin resistance on the same vector as the gene for human insulin production, then any bacteria that are immune to ampicillin will also be able to produce insulin.  

Plasmids

In addition to the main chromosome, some bacteria contain small, accessory rings of DNA called plasmids. 

Bacteria are capable of taking up plasmids from their environment. The genes on the plasmid are then expressed after it is taken up. This process is called transformation because the bacteria have new characteristics; they have been transformed.

Foreign genes can be inserted into plasmids using genetic engineering technology. For example, the gene for human growth hormone has been put in plasmids and taken up by bacteria. The transformed bacteria secrete human insulin.

When the bacteria reproduce, the plasmids are also reproduced. The reproduction of genes that have been added to DNA is called cloning. The genes added to the plasmid have been cloned.

Viruses

Viruses are the vectors of choice for inserting genes into animal cells.

They can accept larger amounts of DNA than plasmids.

When the virus reproduces within the animal cell, it also reproduces the foreign gene that it carries. The gene is therefore cloned.

The cDNA of some retroviruses becomes integrated into the host chromosome.

Restriction enzymes were discovered in bacteria. Bacteria use them as a defense mechanism to cut up the DNA of viruses or other bacteria.

Hundreds of different restriction enzymes have been isolated. Each one cuts DNA at a specific base sequence. For example, EcoRI always cuts DNA at GAATTC as indicated below.

The sequence GAATTC appears three times in the DNA strand below. As a result, EcoRI will cut the strand into four pieces.

Other restriction enzymes cut at different sites, some examples are listed below.

Enzyme	Cutting Site
Bam HI	GGATCC
Hae III	GGCC
Pst I	CTGCAG
Hinf I	GANTC

Sticky Ends

Fragments of DNA that has been cut with restriction enzymes have unpaired nucleotides at the ends called sticky ends. All of the fragments will have the same sticky ends. The sticky ends have complimentary bases, so they could rejoin.

If the vector and the gene to be cloned are both cut with the same restriction enzyme, they will both have complimentary sticky ends.

To make recombinant DNA, restriction enzymes are used to cut DNA from two sources such as the that of a vector and a gene to be cloned. If the vector and the gene to be cloned are both cut with the same restriction enzyme, they will both have complimentary sticky ends.

After cutting, the two samples of DNA are mixed. Some of the fragments from one species will stick to those of the other because they both have the same sticky ends.

DNA ligase is used to seal the fragments.

A genome is all of the genes in a particular organism. Bacteria or virus vectors can be used to store fragments of the DNA from a species.

The DNA is cut up into fragments and the different fragments are inserted into bacteria or viruses. The collection of bacteria or viruses is called a genomic library.

Finding Genes in a Gene Library

Blue-White Screening Method

The plasmid used contains a gene for ampicillin resistance. The transformed bacteria are grown on a medium that contains ampicillin. Any bacteria that grow have been transformed because the bacteria cannot grow unless they are ampicillin-resistant.

The plasmid also contains a gene for the production of b-galactosidase. The b-galactosidase gene contains a region that can be cut with a number of different restriction enzymes. Genes inserted into this site will inactivate the b-galactosidase gene because it has been cut and the new gene has been inserted within it. A recombinant plasmid therefore will not produce b-galactosidase.

The transformed bacteria are grown on a medium that contains X-gal, a substrate for b-galactosidase. Colonies that use X-gal as a food source produce a blue compound. Colonies that have received a gene cannot use X-gal and appear white.

Bacteria that have foreign genes, therefore, will grow (resistant to ampicillin) and appear white (unable to produce b-galactosidase).

Radioactive Probes

The blue-white screening method described above selects for bacteria that have any gene. Radioactive probes can be used to find colonies that have specific genes.

Probes are short, single-stranded segments of DNA whose base sequence matches part of the gene in question. It is not necessary to match the entire gene, just a small fragment.

If a DNA probe is desired, it can be made by first obtaining mRNA for the gene in question. A DNA copy of the gene is made using reverse transcriptase.

When the probe is mixed with the DNA in question, it will form a double-helix in the area where the the gene has complimentary bases.

If the probe is radioactive or fluorescent, it can be visualized. The gene can then be isolated or cloned as needed.

It may be possible to see the chromosome and the location on the chromosome while viewing under a microscope.

Autoradiography is a process in which film is used to show the area of the vector where the probe has attached. This area is the gene in question.

Probes can also be used to detect:

disease-causing microorganisms

defective (disease) genes

various cancers

Eucaryotic genes contain introns but bacteria do not contain the necessary enzymes to remove introns, so eucaryotic genes that are inserted into bacteria must be inserted without introns.

The DNA of eucaryotes is extremely long, containing many thousands of genes. It is often not possible to find specific genes in the DNA. Artificial genes can be made, however, using mRNA.

In order to synthesize a gene, one must first obtain some mRNA produced by the gene in question. Recall that the introns of mature mRNA have already been removed.

Use reverse transcriptase (from retroviruses, see discussion above) to produce a DNA copy of the RNA. This copy is called cDNA.

The polymerase chain reaction can be used to make many copies of small pieces of DNA. Because techniques in biotechnology usually require many copies of genes, PCR has allowed much of the biotechnology development that we have seen in recent years.

Materials needed

The procedure requires primers, DNA polymerase, and nucleotides.

Primers are short chains of about 20 nucleotides that are complimentary to a region in the DNA to be amplified. They are needed because the enzyme that copies the DNA (DNA polymerase) cannot start the process unless it has already been started.

Nucleotides are needed because DNA is composed of nucleotide "building blocks".

Procedure

The DNA in question is heated to separate the two strands of the double helix.

After the strands are separated, the DNA is cooled and the primers attach.

Next, DNA polymerase attaches and copies the strand.

The solution is then heated and cooled at regular intervals. Each time it is heated and cooled, the DNA replication process repeats itself.

In RFLP analysis, the DNA of an organism is cut up into fragments using restriction enzymes. A large number of short fragments of DNA will be produced.

Restriction enzymes always cut at the same base sequence. Because no two people have identical DNA, no two people will have the same length fragments. For example, the enzyme EcoRI always cuts DNA at the sequence GAATTC. Different people are going to have different numbers of this particular sequence and will therefore have different fragment lengths. In addition, some of them will be at different locations on the chromosome.

Electrophoresis is a technique used to separate the DNA fragments according to their size. They are placed on a sheet of gelatin and an electric current is applied to the sheet. DNA is charged and will move in an electric field toward the positive pole.

In the diagram below, holes (wells) in the gelatin can be seen. DNA samples placed in these wells will migrate through the gelatin toward the + side after an electric current is applied.

The smallest fragments will move the fastest because they are able to move through the pores in the gelatin faster. Bands will be produced on the gelatin where the fragments accumulate. The shortest fragments will accumulate near one end of the gelatin and the longer, slower-moving ones will remain near the other end.

In the diagram below, four samples of DNA were placed on the gelatin. After an electric current was applied for a period of time, the fragments separated. Notice that sample D on the right does not match the other three samples.

Uses

This procedure requires a large amount of DNA so it is often used in conjunction with PCR discussed above. Some uses are identification of diseased genes including oncogenes, identification of viral infections, determining family relationships among individuals, and identifying tissue found at a crime scene.

For example, suppose that this procedure is used to identify cells found at a crime scene. Samples A and B (above) came from the scene of the crime and samples C and D came from two different suspects. What can you conclude?

Some genetic diseases that can be identified using this procedure are Sickle Cell disease, Huntington’s disease, Duchenne muscular dystrophy.

Taxonomists can use this technique to explore evolutionary relationships. Individuals of the same species, while not identical, will be more similar than individuals of different species.

The procedure for sequencing and mapping DNA requires RFLP analysis.

Markers

For any restriction enzyme, a chromosome may have many cutting sites, and these vary among individuals but tend to be similar among closely related individuals because their DNA is more similar..

If an investigator wants to identify a gene for a genetic disease, sometimes the gene has a base sequence that causes the DNA to be cut differently than the normal gene. If this is the case, the analysis is performed as described above. People that carry the abnormal gene will produce different fragment lengths because they have different cutting sites.

Usually however, the diseased gene and the normal gene do not differ in their cutting sites. Even though the genes produce the same fragment lengths, it may still be possible to use this technique to determine if an individual has a diseased gene because there may be a nearby cutting site on the same chromosome that the diseased gene (or normal gene) resides. If a cutting site is associated with a certain kind of gene (a diseased gene for example) the presence of the cutting site indicates the presence of the diseased gene. Cutting sites that allow us to identify genes are called genetic markers.

The cutting site has nothing to do with the gene, but because they happen to be located near each other on the same chromosome (closely linked), it can be used. For it to be a reliable indicator, it needs to be very near the diseased (or normal) gene so that crossing-over during meiosis will be unlikely to separate them.

Gene therapy uses technology to change the genetic composition of a cell.

Ex vivo methods are done outside the organism. Cells are removed, treated and returned to the individual.

Retroviruses are often used as the vector. The retroviruses contain recombinant RNA which includes the gene to be added. Once in the cell, the enzyme reverse transcriptase makes a DNA copy of the RNA.

Currently, there are more than 100 clinical trials of this technique.

Example of ex vivo gene therapy

This procedure has been used to treat severe combined immunodeficiency syndrome (SCID). People with this disease are susceptible to infections because their white blood cells do not produce an enzyme needed by their immune systems. This disease has been treated in two different ways. In a short-term solution, the white blood cells were removed and infected with a retrovirus that carried the needed gene. After the cells were replaced, many of the cells contained the gene. White blood cells, however, are short-lived and a long-term solution is to apply this technique to the cells that produce the white blood cells (called stem cells).

In vivo gene therapy treats cells in the individual without removing them.

Retroviruses can be used to introduce genes directly into the body.

Synthetic carriers like liposomes can also be used to carry genes. Liposomes are microscopic lipid vesicles that are readily taken up by cells. If they are coated with DNA, the DNA is also taken up.

Transgenic Bacteria

Protein Products

Many useful human proteins are now synthesized by transgenic bacteria. Some of these are listed below.

Human growth hormone is used to treat dwarfism. It previously took the pituitary glands from over 50 cadavers to make one dose.

Human Insulin is used to treat diabetes.  Insulin was previously obtained from the pancreas of slaughtered cattle and pigs. It sometimes caused allergic reactions.

Tissue plasminogen activator  dissolves blood clots in heart attack victims.

Clotting factor VIII will soon be available. Most cases of hemophilia are due to the absence of this factor.

Human lung surfactant is used in premature infants with respiratory distress syndrome.

Atrial natriuretic hormone can be used to treat hypertension.

Bovine growth hormone (bGH) increases milk production in cows by about 10%.

Vaccines

Vaccines were previously made by killing or weakening a virus or bacteria and then injecting it. Its surface proteins caused an immune reaction. Occasionally, these vaccines would make people ill. Using biotechnology, some of the proteins of the disease organism can be made by cloning the gene that codes for them. These proteins are sufficient to stimulate the immune system but are incapable of causing an infection.

A vaccine for hepatitis B is now produced using biotechnology.

Vaccines for chlamydia, malaria and HIV are being developed.

Vaccines for hoof-and-mouth disease and scours (a form of dysentery) have been developed for farm animals.

Other Uses of Recombinant Bacteria

Bacteria have been produced that inhibit the formation of ice crystals. These bacteria have been released onto crop plants to protect them from frost damage.

A bacteria species that normally colonize corn roots have been given a gene that enables it to produce an insect-killing toxin (pesticide).

Bacteria are being developed that do a better job at breaking down oil. These may be useful to help clean up oil spills.

Bacteria have been developed that are capable of removing some kinds of toxins from the air and water.

Bacteria have been engineered to extract metals from low-grade ore (bioleaching). This technique is currently being tested..

Transgenic plants

The only plasmid that plant cells take up is from the bacterium Agrobacterium. Not all plants take up the plasmid.

It may be difficult to create transgenic plants because plants have a cell wall. One solution is to remove the cell wall. These cells (called protoplasts) are then placed in a liquid with foreign DNA. An electric current is used to make small, temporary holes in the membrane and allow the DNA to pass in.

Presently, there are approximately 50 types of genetically engineered plants that resist insects, viruses, and herbicides. Field trials have begun on these plants.

A weed called mouse-eared cress has been designed to produce a biodegradable plastic called polyhydroxubutrate (PHB).

In the future, biotechnology may be able to improve crop yields and produce plants that contain all of the amino acids required for human consumption. If plants could produced that can fix atmospheric nitrogen, they would require considerably less fertilizer. Plants engineered to grow under harsh environmental conditions could allow wastelands to be more productive.

Transgenic Animals

Animal cells generally will not take up plasmids. Other methods such as microinjection must be used.

One method has been developed where animal eggs are placed in a mixer with needle-like fragments of silicon carbide. The needles make holes in the cells, allowing DNA to enter. Using this procedure, eggs from fish and several agricultural species have been given the gene for bovine growth hormone, producing larger individuals.

Pharmaceutical companies are developing techniques to produce chemicals using animals. The drug is produced in the milk of females. For example, goats have been developed to produce antithrombin III, used to prevent blood clots. Clinical trials of this drug will begin soon.

Lactoferin is added to infant formula to transport iron and to prevent bacterial infections in the gastrointestinal tract. A transgenic bull has been produced that carries a gene for the production of human lactoferin. Females will produce milk with lactoferin.

A sheep has been bioengineered to produce tPA (tissue plasminogen activator) in her milk.

A pig has been produced that can produce human hemoglobin. Artificial blood may soon be a reality.

Cloning Mammals

Cloning animals refers to producing offspring that are genetically identical to the animal being cloned. This process has been done by removing the nucleus of an egg and replacing it with a diploid nucleus from the organism to be cloned. The egg is then treated so that it begins dividing. It is placed in the uterus of a host animal where it continues to grow.

The Human Genome Project is a massive, government-funded project whose goal is to determine the base sequence of all of the human chromosomes.

It would take 200 volumes of 1,000 pages each just to list the letters of the bases.

Once the sequences of all genes are know, it will be easier to study, diagnose, and treat many kinds of human genetic diseases. In addition, it may make it possible to treat more diseases using gene therapy techniques.

The information is expected to lead to a better understanding of genetic systems, and ultimately answers to mysteries surrounding such topics as gene regulation and cancer.