gene cloning-the polymerase chain reaction Flashcards
What is DNA polymerase?
- an enzyme capable of joining together tens of thousands of nucleotides in a matter of minutes
- one such enzyme, taq polymerase, is obtained from bacteria in hot springs and is therefore tolerant to heat (thermostable) and does not denature during the high temperatures used as part of the process
What is a thermocycler?
-a computer-controlled machine that varies temperatures precisely over a period of time
What are the advantages of in vitro gene cloning?
- it is extremely rapid: within a matter of hours 100 billion copies of a gene can be made
- this is particularly valuable where only a minute amount of DNA is available, for example, at the scene of a crime
- this can quickly be increased using the polymerase chain reaction and so there is no loss of valuable time before forensic analysis and matching can take place
- a complicating factor is that PCR will also increase massively any other contaminating DNA found at the scene
- in vivo cloning would take many days or weeks to produce the same quantity of DNA
- it does not require living cells: -all that is required is a base sequence of DNA that needs amplification
- no complex culturing techniques, requiring time and effort, are needed
What are the advantages of in vivo gene cloning?
- it is particularly useful where we wish to introduce a gene into another organism: -as it involves the use of vectors, once we have introduced the gene into a plasmid, this plasmid can be used to deliver the gene into a plasmid, this plasmid can be used to deliver the gene into another organism, such as a human being (i.e. it can transform other organisms)
- this is done in a technique called gene therapy: -this is because a gene that has been cut by the same restriction endonuclease can match the sticky ends of the opened-up plasmid
- contaminant DNA will therefore not be taken up by the plasmid
- in vitro cloning requires a very pure sample because any contaminant DNA will also be multiplied and could lead to a false result
- it involves almost no risk of contamination -they DNA copies has few, if any, errors
- at one time, about 20% of the DNA cloned in vitro by the PCR was copied inaccurately, but modern techniques have improved the accuracy of the process considerably
- however any errors in copying DNA or any contaminants in the sample will also be copied in subsequent cycles
- this problems hardly ever arises with in vivo cloning because, although mutations can arise, these are very rare
- it is very accurate: -they DNA copies has few, if any, errors
- at one time, about 20% of the DNA cloned in vitro by the PCR was copied inaccurately, but modern techniques have improved the accuracy of the process considerably
- however any errors in copying DNA or any contaminants in the sample will also be copied in subsequent cycles
- this problems hardly ever arises with in vivo cloning because, although mutations can arise, these are very rare
- it cuts out specific genes: -it is therefore a very precise procedure as the culturing of transformed bacteria produced many copies of a specific gene and not just copies of the whole DNA sample
- it produced transformed bacteria that can be used to produce large quantities of gene products: -the transformed bacteria can produce proteins for commercial or medical use (e.g. hormones such as insulin)
What are the benefits of recombinant DNA technology?
1-microorganisms can be modified to produce a range of substances, for example, antibiotics, hormones and enzymes, that are used to treat diseases and disorders
2-microorganisms can be used to control pollution, for example, to break up and digest oil slicks or destroy harmful gases released from factories
-care needs to be taken to ensure that such bacteria do not destroy oil in places where it is required, for example, car engines
-to do this, a suicide gene can be incorporated that causes the bacteria to destroy themselves once the oil slick has been digested
3-genetically modifies plants can be transformed to produce a specific substance in a particular organ of the plant
-these organs can then be harvested and the desired substance extracted
-if a drug is involved, the process is called plant charming
-one promising application of this technique is in combating disease
-this involved the production of plants that manufacture antibodies to pathogens and the toxins they produce
-alternatively the plants can be modified to manufacture antigens which, when injected into humans, induce natural antibody production
4-genetically modified crops can be engineered to have financial and environmental advantages
-these include making plants more tolerant to environmental extremes, for example, able to survive drought, cold, heat, salt, or polluted solid, etc.
-this permits crops to be grown commercially in places where they do not grow at present
-globally, each year, an area of land equal to half the united kingdom becomes unfit for normal crops because of increases in soil salt concentrations
-growing of genetically modified plants, such as salt-tolerant tomatoes, could bring this land back into productivity
-in a world where millions lack a basic nutritious diet, and with a predicted 90 million more mouths to feed by 2025, can we ethically oppose the use of such plant crops?
5-genetically modified crops can help prevent certain diseases
-a type of rice, called golden rice, can have a gene for vitamin A production added
-can we justify not developing more vitamin A-enriched crops when 250 million children worldwide are at risk from vitamin A deficiency leading to 500000 cases of irreversible blindness each year?
6-genetically modified animals are able to produce expensive drugs, antibiotics, hormones and enzymes relatively cheaply
7-replacing defective genes (gene therapy) might be used to cure certain genetic disorders, such as cystic fibrosis and severe combined immunodeficiency
8-genetic fingerprinting can be used in forensic science
What are the risks of recombinant DNA technology?
1-it is impossible to predict with complete accuracy what the ecological consequences will be of releasing genetically engineered organisms into the environment
-the delicate balance that exists in any habitat may be irreversibly damaged by the introduction of organisms with engineered genes
-there is often no going back once an organism is released although suicide genes can be inserted or the organism engineered so it can only survive when a supplement is added
2-a recombinant gene may pass from the organism it was placed in, to a completely different one
-we know, for example, that viruses can transfer genes from one organism to another
-what is a virus were to transfer genes from organism to another
-what is a virus were to transfer genes for herbicide resistance and vigorous growth from a crop plant to a weed that completed with a crop plant?
-what is the same gene were transferred in pollen to other plants?
-how would we then be able to control this weed?
3-any manipulation of the DNA of a cell will have consequences for the metabolic pathways within that cell
-we cannot be sure until after the event what unforeseen by-products of the change might be produced
-could these lead to metabolic malfunctions, cause cancer, or create a new form of disease?
4-genetically modified bacteria often have antibiotic resistance marker genes that have been added
-these bacteria might spread antibiotic resistance to harmful bacteria
5-all genes mutate
-what then, might be the consequences of our engineered gene mutating?
-could it turn the organism into a pathogen which we have no means on controlling?
6-what will be the long-term consequences of introducing new gene combinations?
-we cannot be certain of the effects on the future evolution of organisms
-will the artificial selection of desired genes reduced the genetic variety that is so essential to evolution?
7-what might be the financial consequences of developing plants and animals to grow in new regions?
-developing bananas which grow in Britain could have disastrous consequences for the Caribbean economies that rely heavily on this crop for their income
8-how far can we take the technique of replacing defective genes?
-it may be acceptable to replace a defective gene to cure cystic fibrosis, but is it equally acceptable to replace a defective gene to cure cystic fibrosis, but is it equally acceptable to introduce genes for intelligence, more muscular bodies, cosmetic improvements, or different facial features?
9-will knowledge of, and ability to change, human genes lead to eugenics, whereby selection of genes leads to a means of selecting one race rather thank another?
10-what will be the consequences of the ability to manipulate genes getting into the wrong hands?
-will unscrupulous individuals, groups or governments use this power to achieve political goals, control opposite or gain ultimate power?
11-is the financial cost of recombinant DNA technology justified, or would the monkey be better used fighting hunger and poverty, that are the cause of much human misery
-will sophisticated treatments, with their more high-profile images, be put before the everyday treatment of rheumatoid arthritis or haemorrhoids?
-will such treatments only be within the financial reach of the better-off?
12-genetic fingerprinting, with its ability to identify an individual’s DNA accurately, is a highly reliable forensic tool
-how easy would it be for someone to exchange a DNA sample maliciously, leading to wrongful conviction?
13-how do we deal with the issues surrounding the human genome project?
-is it right that an individual or company can patent, and therefore effectively own, a gene?
What is the treatment of servers combines immunodeficiency using gene therapy?
- servers combined immunodeficiency (SCID) is a rare inherited disorder
- people with this condition do not show a cell-mediated immune response, nor are they able to produce antibodies
- the disorder arises when individuals inherit a defect in the gene that codes for the enzyme adenosine deaminase (ADA)
- this enzyme destroys toxins that would otherwise kill white blood cells
- survival has depended upon patients being given bone marrow transplants and/or injections of ADA
What are the recent attempts to treat the disorder using a technique called gene therapy?
- the normal ADA gene is isolated from healthy human tissue
- the ADA gene is inserted into a retrovirus
- the retroviruses are grown with host cells in the laboratory to increase their number and hence the number of copies of the ADA gene
- the retroviruses are mixed with the patient’s T cells into which they inject a copy of the normal ADA gene
- the T cells are reintroduced into the patient’s blood to provide the coded information needed to make ADA
