Biotechnology

What Is Biotechnology?• 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.

Tools of the Trade …

• Viruses Structure

Reproduction

viral DNA mRNA protein

viral RNA cDNA mRNA protein

Recombinant DNA TechnologyVectors

• Vectors are DNA used to transfer genes into a host cell.

• A vector must be capable of self-replicating inside a cell.

• Marker genes can be used to determine if the gene has been taken up.

SEE AMPICILLIN

Recombinant DNA Technology Plasmids

• The host bacterium takes up the plasmid, which includes the foreign gene.

• When the bacteria reproduces, the plasmids are also reproduced. The gene is cloned.

• Shuttle vectors are plasmids that are capable of existing in several different species. They are useful when transferring genes to multicellular organisms.

Recombinant DNA TechnologyViruses

• Viruses are the vectors of choice for 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 DNA of some retroviruses becomes integrated into the host chromosome.

Recombinant DNA TechnologyRestriction enzymes

• 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.

Other examples

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

Sticky Ends

BLUNT ENDS??

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

Making Recombinant DNA

• COMPLETE THE RECOMBINANT DNA ACTIVITY

Genomic Libraries

• 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.

Other Methods of Delivering DNA

• Electroporation involves using an electric current to create pores in the cell wall and plasma membrane for DNA to enter.

• It is difficult to create transgenic plants because the cell wall prevents entry of DNA. One solution is to remove the cell wall. These cells (called protoplasts) are then placed in a liquid with foreign DNA. Electroporation is a technique that uses an electric current to make small, temporary holes in the membrane DNA can pass in.

• A gene gun propels small gold pellets coated with DNA. The pellets penetrate the cell wall and plasma membrane and enter the cell to deliver their DNA.

Polymerase Chain Reaction (PCR)

• 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 and Procedure

short chains of about 20 nucleotides

Complementary to DNA Gene

from the thermophile Thermus aquaticus is used because this species thrives at temperatures that are near boiling.called Taq polymerase

Procedure

• 1) The DNA in question is heated to approximately 95 degrees C to separate the two strands of the double helix.

• 2) After the strands are separated, the DNA is cooled to about 50 degrees and the primers attach.

• The temperature is raised to approximately 70 degrees C. This is the optimal temperature for Taq polymerase. The polymerase attaches to 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.

DNA Fingerprinting (RFLP Analysis)

• 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.

• 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.

GEL-E LABS + RFLP ACTIVITIE

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.

CONT’D

The DNA bands must be stained to make them visible. Ethidium bromide-stained DNA will fluoresce when illuminated with UV light.PCR techniques are used to produce sufficient quantities of DNA for this technique.

Uses for Electrophoresis

• identification of diseased genes including oncogenes,

• identification of viral infections, • determining family relationships among

individuals,• and identifying tissue found at a crime scene.• Taxonomists can use this technique to explore

evolutionary relationships.

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

• Ex vivo• Ex vivo methods are done outside the organism. Cells

are removed, treated and returned to the individual.• In vivo• In vivo gene therapy treats cells in the individual

without removing them.• Retroviruses can be used to introduce genes directly

into the body.

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).

Products Made Using Biotechnology

• Genes that code for the desired protein can be inserted into plasmids and the plasmids are used to transform cells. 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.

CONT’D

• 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%.

BESIDES BACTERIA …

• Vaccines• Transgenic plants• Transgenic Animals• Cloning Mammals

Safety and Ethical Issues?