Unit 18: Genetics and Genetic EngineeringAssignment title: Applications of Genetic EngineeringName: Hani OladeindeStudent I.D: OLA17005525Task 1InsulinA lot of insulin can be made easily and cheaply. There is no risk of insulin being contaminated with a virus or other diseases. There are no ethical issues such as giving people of certain religious faiths material from a pig. No one raising ethical objections about bacteria being exploited. These bacteria are cultured in large vessels with controlled conditions they can be grown anywhere in the world.
The bacteria in question are also modified so that they lack an enzyme and cannot make some of the nutrients they need. So they can only survive in the laboratory environment where they are given such nutrients. They cannot escape into the environment and survive there. There are strict guidelines for people working with these organisms to minimise any risk of contamination or any other potential biohazards.Human growth hormonesSome children grow at a slower rate than normal because their anterior pituitary glands do not make enough human growth hormone.
This condition can be treated by injecting the children with human growth hormone. This is used to be obtained from the pituitary glands of corpses but there have been cases of new variant Creutzfeldt-Jakob disease which is caused by and transmitted by an abnormal protein called a prion protein being transferred to some recipients of the hormone. Genetically modified bacteria can now be used to make human growth hormone. It is safer as there is no risk of transmitting CJD. It is easier to produce human growth hormone in large quantities so all children with pituitary dwarfism can be treated. It is cheaper to produce and may be more acceptable for those being treated.Task 3Restriction digestion is a process in which DNA is cut at specific sites, dictated by the surrounding DNA sequence. Restriction digestion is accomplished by incubation of the target DNA molecule with restriction enzymes that recognize and bind specific DNA sequences and cleave at specific nucleotides either within the recognition sequence or outside of the recognition sequence. Restriction digestion is usually used to prepare a DNA fragment for subsequence molecular cloning as the procedure allows fragments of DNA to be pieced together like building blocks through ligation.Steps of recombinant DNAIsolation of genetic materialThe first step in recombinant DNA technology is to isolate the desired DNA in its pure form. Lysozyme is used to break the bacterial cell wall, cellulose is used to break the plant cell wall, chitinase is used to break the fungal cell wall, ribonuclease removes the RNA and protease the proteins. The addition of ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified DNA.Restriction enzyme digestionRestriction enzymes act as molecular scissors that cut DNA at specific locations. They involve the incubation of the purified DNA with the selected restriction enzyme, at conditions optimal for that specific enzyme.Amplification using PCRPolymerase Chain Reaction is a method of making multiple copies of a DNA sequence using the enzyme, DNA polymerase. It helps to amplify a single copy or a few copies of DNA into thousands to millions of copies. The cut fragments of DNA can be amplified using PCR and then ligated with the cut vector.Ligation of DNA moleculesThe purified DNA and the vector of interest are cut with the same restriction enzyme. This gives us the cut fragment of DNA and the cut vector that is now open. Ligation is the process of joining these two pieces together using the enzyme DNA ligase. The resulting DNA is recombinant DNA.Applications of recombinant DNARecombinant DNA technology is used to make microbes, plants, and animals that carry genes from other species. Recombinant DNA technology can be used in the prenatal diagnosis of human genetic disease.Development of Transgenic Plants:Development of Root Nodules in Cereal CropsProduction of AntibioticsProduction of Hormone InsulinProduction of EnzymesGel electrophoresisGel electrophoresis separates DNA fragments that are of different lengths. First the DNA sample to be analysed is incubated at 35oC for about an hour with a restriction endonuclease enzyme. Restriction enzymes are obtained from bacterial cells. Bacteria use them to cut DNA to protect themselves from invading viruses. Each restriction enzyme cuts length of DNA at a specific base pair sequence called a restriction site. They break bonds between sugar and phosphate groups in the DNA backbone.The restriction enzyme used will recognise a specific base sequence in a length of DNA and make a cut wherever that sequence is. This produces DNA fragments of varying length.Once the DNA has been cut by the restriction enzymes bromophenol blue is added to make the DNA solution denser.The electrophoresis tank is set up and liquid agarose gel is poured into it. There are combs in place and once the gel has set these are removed leaving wells in one end of the gel.A buffer solution is poured into the tanks so that the gel is covered.The DNA samples to be analysed or compared are each added to a well. This has to be done carefully. The micropipette is held above the well and the dyed solution allowed to fall into the well.A cathode and anode are placed at both ends of the tank with the anode at the end away from the wells. A power supply is connected to them and left for about two hours.Because DNA has an overall negative charge because of its many phosphate groups, the fragments travel through the gel towards the anode which is positive.Shorter fragments of DNA travel further than longer fragments. After 2 hours, the power supply is disconnected and the buffer solution is poured away. A dye is added and this strains the DNA fragments to show the resulting banding pattern.Applications of gel electrophoresisIn the separation of DNA fragments for DNA fingerprinting to investigate crime scenes.To analyse results of polymerase chain reaction.To analyse genes associated with a particular illness.In DNA profiling for taxonomy studies to distinguish different species.In paternity testing using DNA fingerprinting.In the study of structure and function of proteins.In the analysis of antibiotic resistance.In blotting techniques for analysis of macromolecules.In the study of evolutionary relationships by analysing genetic similarity among populations or species.Task 4Producing InsulinmRNA is obtained from cells in human pancreas tissue that make insulin. The enzyme reverse transcriptase obtained from some viruses is used to make a single complementary strand of DNA from this mRNA.The enzyme DNA polymerase is then used to make the other complementary DNA strand. Now we have the gene for insulin which is a length of double-stranded DNA that has the code for making the protein insulin.Three unpaired nucleotides are put onto both ends of the molecule. These unpaired nucleotides are called sticky ends. Plasmids are obtained from E. coli bacteria.A restriction enzyme is used to cut open the plasmid. The enzyme makes a staggered cut and leaves sticky ends. The sticky ends that are added to the insulin gene are complementary to the plasmid sticky ends.Plasmids and genes for insulin are mixed in an Eppendorf tube and DNA ligase enzyme is added. Complementary sticky ends can join by hydrogen bonds and DNA ligase catalyses the condensation reaction between the sugars and phosphates in the DNA backbones.The plasmid containing a human insulin gene is called recombinant DNA. The recombinant plasmids and E. coli bacteria are mixed. Calcium chloride is added and they are subjected to heat shock. Some bacteria take up recombinant plasmids and these will be capable of making human insulin.Some of the E. coli bacteria will take up plasmids. However, some won’t take up plasmids and some will take up a plasmid that does not have an insulin gene in it. The bacteria have to be screened to find out which ones are transformedThe antibiotic resistance genes on the E. coli plasmids are used as maker genes. The plasmids are cut at a restriction site that is in the middle of a gene for resistance to tetracycline. If the plasmid accepts a human insulin gene then the tetracycline resistance gene will not work. However it still has an intact gene for resistance to ampicillin.The bacteria are plated out onto agar plates containing ampicillin. Any bacteria that didn’t take up a plasmid won’t grow. Those that have taken up a plasmid will grow and form visible colonies. However, we need to know which have taken up recombinant plasmids.A sterile piece of velvet is placed on the surface of the agar plate that contains tetracycline. This transfers bacteria from colonies on one plate to the other plate. On the tetracycline plate only colonies that have a plasmid but no human insulin gene will grow.So now you know which colonies on the ampicillin plate you need. These bacteria are taken and grown in large culture vessels and their insulin is harvested.