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Gene technology
The document outlines the steps involved in genetically engineering bacteria to produce human insulin. These include identifying and obtaining the insulin gene from human pancreas cells, inserting the gene into a bacterial plasmid vector, transforming the vector into host bacteria, selecting transformed bacteria using antibiotic resistance markers, and cloning the bacteria to mass produce human insulin. The document also discusses some advantages of producing insulin through genetic engineering compared to extracting it from animals.
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Gene technology
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- 2. STEPS INVOLVED INTHE GENETIC ENGINEERING OF BACTERIA TO SYNTHESIZE HUMAN INSULIN. IDENTIFYING & OBTAINING THE GENE REQUIRED ◦ The gene for insulin is active in the B-cells of the pancreas. ◦ If a gene is active it usually produces thousands of mRNA molecules which are complementary to the gene. ◦ Therefore the mRNA is isolated from the B cells of the pancreas. ◦ Reverse transcriptase is used to make cDNA and DNA polymerase clones the DNA. ◦ Restriction enzymes/restriction endonucleases cut the DNA at specific base sequences (restriction site). ◦ They leave sticky ends (short lengths of unpaired bases). PUTTING THE GENES INTO A VECTOR ◦ The same restriction enzymes that were used to cut the donor should be used to cut the plasmid DNA leaving sticky ends. ◦ The wanted genes are mixed together with the plasmid DNA and they join up at the sticky ends. ◦ The initial attraction is due to hydrogen bonding. ◦ DNA ligase ensures that the gene is permanently added to the plasmid forming recombinant DNA.
- 3. INTRODUCING VECTOR DNAINTO THE HOST CELL ◦ The plasmids containing the human insulin gene are mixed together with the bacterial cells. ◦ Those bacteria that take up the plasmids are said to be transformed. CLONING THE DNA ◦ The bacteria that are transformed are cloned to produce genetically identical offspring. ◦ Every time a bacterium divides, it will replicate the human insulin gene. SELLECTING THE TRANSFORMED BACTERIA ◦ Once the plasmid and DNA are mixed, not all the bacteria are transformed and not all the plasmid will have taken up the foreign DNA. ◦ This can be avoided by using: A gene resistant to a particular antibiotic-If the bacteria are grown on a medium containing that antibiotic, only the transformed ones will multiply and form colonies. The DNA that survive growing on the antibiotic are then grown on X-gal and those that do not contain the donor DNA appear blue but those that contain it will appear colourless and they can be isolated for further cloning.
- 4. ADVANTAGES OF TREATINGDIABETICS WITH HUMAN INSULIN PRODUCED BY GENE TECHNOLOGY. Back in the day before bacteria were used to produce human insulin gene, people with insulin dependent diabetes were injected with insulin from pigs or cattle. Pig or cattle insulin is similar to human insulin but it is not identical. ADVANTAGES It is chemically identical, so there is little chance of an immune response. It is an exact fit in the human insulin receptors in human cell surface membranes, hence causing rapid response. Like in human insulin, the response is much shorter than the cow or pig insulin. It overcomes problems related to the development of tolerance to cow or pig insulin. It avoids any ethical issues, such as religious objections or from vegetarians .
- 5. WHY PROMOTERS NEEDTO BE TRANSFERRED ALONG WITH THE DESIRED GENES. Not all the genes in a cell are switched on at any one time Certain regions of DNA called promoter regions are next door to the genes that have to be activated before a gene is expressed. If a human insulin gene is transferred in a prokaryote DNA without adding the required prokaryote promoter, it will not be transcribed and hence will not be expressed. The promoter initiates transcription of the gene so that the desired product is expressed.
- 6. WHY FLOURESCENT MARKERSOR (EASILY STAINED SUBSTANCES) ARE NOW USED INSTEAD OF ANTIBIOTIC RESISTANT MARKERS. HOW ARE ANTIBIOTIC MARKERS USED? Not all the bacteria are transformed and there needs to be a way to identify those which have taken up the plasmids and those that have not. The first methods were based on antibiotic resistant markers. The method used to identify the bacteria with the required DNA recombinant IS: ◦ ◦ ◦ The original selected plasmid has antibiotic resistance genes to two antibiotics A and B and those bacteria that have the plasmid will grow successfully in the presence of these antibiotics but those lacking will be killed. Restriction enzymes are used to cut the middle of one of the antibiotic resistance gene, B. Those bacteria that had been transformed will no longer have a working copy of antibiotic B as a result of being interrupted by the cDNA insulin gene. Many plasmids have a working copy of the antibiotic resistance gene to antibiotic B, showing that the plasmids have failed to form recombinant DNA. However those that have taken up the recombinant DNA do not have a working copy of antibiotic A and there are ways to identify them. This is:
- 7. The bacteriaare spread on an agar plate containing antibiotic A and those that have taken up the plasmid with recombinant DNA survive. A sponge is used to touch briefly on the agar, picking up some bacteria and these are touched onto a sterile agar plate containing antibiotic B and those that have the recombinant DNA are killed by this antibiotic so that their original location on the plate is known with the help of the original agar plate that had been refrigerated. One potential problem of using antibiotic markers in this way is that: The antibiotics are present on the plasmids meaning that if these genetically engineered bacteria come into contact with pathogenic bacteria, the resistance genes can easily be transferred to them making it very difficult to control the spread of such bacteria. Fluorescent markers are quicker and produce a higher yield proportion of transformations.
- 8. THE BENEFITS ANDHAZARDS OF GENE TECHNOLOGY BENEFITS It is fast It is possible to produce genetically engineered organism for a specific purpose. It involves unrelated organisms Specific products are got, avoids dependence on cows and pigs. Reduce use of herbicides and pesticides Can be used to treat CF as well as in cancer treatment. HAZARDS Transfer of genes inserted, to other bacterial species can occur causing resistance Crops may end up producing wild relatives(Mexican wild maize) Food may trigger toxic or allergic reactions when consumed.
- 9. THE SOCIAL ANDETHIAL IMPLICATIONS OF GENE TECHNOLOGY SOCIAL Enhance crop yields(crops can grow outside normal location and season) Enhance nutritional content of food. Leads to effective and cheaper medicines through gene manipulation. Produce supper weeds. Increases costs of seeds Reduce crop biodiversity by out competing natural crops Causes antibiotics to become less useful and causes allergic reactions ETHICAL It is good to conduct such as technological improvements are done. It may be wrong as some aspects of it may not be understood. It is wrong as some organisms may escape and pause risks. It is wrong as it cannot be reversed and future implications are unknown.
- 10. THE USE OFELECTROPHORESIS IN GENETIC FINGERPRINTING AND DNA SEQUENCING. ELECTROPHORESIS It is a method of separating and analyzing molecular structure based on the rate of movement of fragments of DNA in a liquid medium while under the influence of a magnetic field. The type we use is the gel one. The gel has spaces between it and the molecules can move freely under the influence of a magnetic field. Electrodes are placed on either side of the gel. Restriction enzymes are used to cut the DNA into fragments of different lengths. The direction of movement is due to the DNA molecules being negatively charged hence move to the anode. The distance moved in a given time will depend on the size of the molecule/fragment. When DNA is cut by restriction enzymes, the fragments are similar but not exactly the same. As DNA is transparent and invisible we have to: Stain the DNA with methyl blue. Create a gene probe: this is a single stranded DNA molecule with a base sequence complementary to the DNA you wish to identify. To identify where the DNA has attached itself, the probe must be labeled by: ◦ Making the probe radioactive ◦ Staining the probe with fluorescent stain eg. Vital red
- 11. GENETIC FINGERPRINTING Once theDNA fragments have been separated by gel electrophoresis they can be compared with other samples of DNA, hence allowing the determination of the source of DNA. DNA SEQUENCING The best example is the genome project. Electrophoresis is used to separate DNA fragments to enable the determination of the order of the bases within the genes and chromosomes.
- 12. THE CAUSES ANDSYMPTOMS OF CYSTIC FIBROSIS CAUSES It is caused by the gene coding for the transmembrane protein that transports chloride ions through the cell surface membrane. Its inheritance is autosomal and recessive. The gene is located on chromosome 7. EFFECTS OF CF Reduced chloride transport-which leads to the production of thick, sticky mucus which affects the lungs, pancreas and reproductive organs. The mucus blocks the pancreatic duct preventing amylase and protease enzymes from reacting in the ileum, hence affecting digestion and nutrition. The mucus remains there and leads to wheezing and repeated infections. The mucus may block the sperm ducts.
- 13. PROGRESS TOWARDS TREATINGCF WITH GENE TECHNOLOGY Since it is the mucus in the lungs that limits life span, it is these cells that have been the focus of efforts. It is thought that if even a small proportion of lung cells could be given a working copy of the cells that would thin the mucus sufficiently. For CF, a vector must be use to deliver the DNA containing the functional CFTR gene. They could use vectors such as: Viral delivery system- using viruses, the intension being to infect the lung surface cells which releases the genetic material into the cells where it is expressed. Non-viral delivery systems Gene therapy has not been successful yet because: ◦ Current viral vectors have been found to cause infections. ◦ Some have been found inefficient. ◦ The effect of therapy only lasts a few days.
- 14. GENETIC SCREENING ANDCOUNCELLING The pattern of inheritance varies according to whether the allele is dominant, recessive or sex-linked. GENETIC SCREENING It is the testing of a sample of DNA from a group of people to identify the presence or absence of particular alleles. Such screening may be: Carrier screening- potential parents may be screened; all individuals in a family may be screened as a result of one member of the family developing a genetic condition. Prenatal screening-screening the genetic makeup of an unborn child to detect genetic conditions. New-born screening-screened for genetic conditions to maintain stability. Genetic conditions that may be screened for are: ◦ Chromosomal abnormalities- Down’s syndrome, trisomy 13 and 18. ◦ Single gene disorders- haemophilia, sickle ell anaemia and cf. ◦ Neural tube defects- spina bifida and anencephaly. Once the results of a genetic test are known it is necessary for those involved to receive genetic counseling.
- 15. GENETIC COUNCELLING It isrequired to give an explanation of the results, probabilities, dangers, diagnosis and treatment. For the individual and for couples who are aspiring to have children. ETHIAL CONSIDERATIONS Who decides who to be screened or tested? Which specific disorder should be screened? Who should be providing the screening? Should we screen for disorders that have no cure? Should there results be confidential? If not should they have access to the information? Or should they be made available to potential employers, insurers etc.