BIOTECHNOLOGY Biotechnology is the process by which living organisms are used to create new products THE ORGANISMS Bacteria: are prokaryotic organisms that contain circular DNA and no organelles. They are classified in the following way: Gram Negative: Bacteria contains three layers: an outer membrane, a peptidoglycan layer and a cytoplasmic membrane. This makes it tougher and more resistant. They would look pink or light purple when seen under microscope. Gram Positive: Gram-positive bacteria cell wall is much thicker, lacks the cell envelope and it contains additional substances such as teichoic acids, polymers composed of glycerol. 1
Viruses, Bacteriophages: Viruses or bacteriophages are proteins packages that contain DNA molecules. This molecules infect bacteria, hence their name bacteriophage (bacteria eater) Yeast: Eukaryotic organisms that could be used to represent human cell to a limited extent. Human/ mammals cells: These cells could be extracted from patients, treated and used to restore equilibrium. TECHNIQUES DNA Sequencing Large-scale DNA sequencing uses the dideoxy method with some improvements for increased efficiency and speed. The use of fluorescently tagged dideoxynucleotides ena1;>les all four reactions to occur in one tube, and the sequence can be read by machines. DNA fragments are separated on mass-produced capillary gels. DNA sequencing This method was developed by a British scientist Frederick Sanger. It is used to determine the nucleotide sequence of DNA molecules. This technique was used by scientists to map the human genome. This method used a modified nucleotide, which terminates the growing DNA chain. Procedure: 1. 4 tubes (portions) containing the DNA strand to be prepared 2.Each portion is given all the ingredients needed for DNA synthesis and in addition, each contains one of the four nucleotides in the modified form 3.Synthesis of new strand is allowed to take place 4.the new DNA strand from the 4 portions are separated by electrophoresis 5.the sequence of newly form strand can be read from the autoradiograph (x-ray picture). This is because by reading what the longest strand terminates with, we would know the base of the first nucleotide. The second longest strand will tell us the base of the second nucleotide and so on. 2
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DNA amplification (from Chapter 12.3) Polymerase Chain Reaction (PCR) invented in the early 1980 s by Kary B. Mullis. PCR is one of the most important advances in molecular biology. The procedure requires the making of specific primers and a thermo-stable enzyme called Taq polymerase. First the DNA is heated so the strands are denatured (detach from one another) Then it is allowed to cool so primers would anneal (attach) to the DNA. The reaction is heated again to about 72 C which is the optimal temperature for Taq polymerase to work, hence duplicating the sequence of DNA 4
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Southern Blotting It is used to find or probe specific sequences of DNA. There are also Northern (RNA) and Western (Protein) blotting techniques. 7
DNA fingerprinting These are specific patterns of bands which is of forensic use, because the probability that two people have the same banding patterns would be very small. It is commonly used for crimes and paternity cases. 8
DNA MANIPULATION Transformation.- the uptake foreign plasmid DNA by bacteria Restriction Enzymes- Enzymes that cut specific DNA sequences (e.g.ecori). They usually preferred pallindromic sequences. Plasmids and Vectors- These are small circular pieces of DNA could be used to carry artificially or naturally manufactured DNA. Expression vectors- vectors that have a promoter region just before the added fragments. Expression markers allows cdna- DNA fragments used that have no introns Gene libraries- Cut entire genomes of different organisms using restriction enzymes, you will obtain little fragments of DNA which could be incorporated into vectors, then transform bacteria with it. Cloning- When cloning, you will take your plasmid or vector and insert it in bacteria. Then allow the bacteria to divide or multiply. Restriction Fragment length Polymorphism (RFLP)- DNA segments resulting from restriction enzymes, useful for genetic markers. The probability that 2 persons would have the same sequence in the same place of their genome is 1/6,000,000,000. Used in the O.J.Simpson's case. USEFUL APPLICATIONS Mapping the Human Genome officially begun in 1990, they have done a genetic linkage, physical mapping and DNA sequencing. In June 2000, the US Human Genome Project and Celera Genomics Corporation finish the DNA sequence of the human genome. The next step of this project will take a couple of more years to complete. This completion will NOT come with controversy since they are trying to decipher the DNA code. Human Gene Therapy.- someday this will be a way to correct genetic disorders. There has been no proven benefits to human patients. This techniques involves the use of viruses, bacteria and recombinant techniques. 9
Pharmaceutical products.- Many pharmaceutical products now are being produced by the use of recombinant DNA techniques. Biotech companies can patent materials, methods and products so they cannot be duplicated by anyone. Their best interest is the health of society or is it? In 1996 Genentech filed a lawsuit against Amgen for a drug called Neupogen which help cancer patients with side effects of chemotherapy. Forensics use.- DNA techniques are now widely used to associate suspects with crimes. These techniques have help many innocent people get out of jail. Environmental use- scientists are trying to find ways to engineer bacteria to do the dirty work. Non-biodegradable materials could be and perhaps will be degraded by bacteria in the future. Agricultural use- agricultural plants have been tweaked for centuries to perform better than regular plants. In the past decades, we have been able to manipulate their DNA to perform even better. We now have plants that are bug-resistant, last longer and are bigger. Animals are also being manipulated so they can produce more than expected. REGULATORY AGENCIES Center for Disease Control CDC investigate pathogens and non pathogens around the country. They work will ALL levels of bacteria from E.coli to Ebola. Food and Drug Administration FDA Control the types of food that we are exposed to. They also control the biotechnology industry (pharmaceutical and medical device) from this country and others. International Standard Organization (ISO) control the European biotechnology industry that supply any products to Europe. 10