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Abstract
This is artificial manipulation of living organisms’ genome and genotypes by use of various chemicals. After manipulation, the generated phenotypes of the organisms are engineered by use of recombinant DNA technology.
Polymerase chain reaction (PCR)
This is an artificial method of duplicating or generating similar number of copies of a DNA strands into large numbers. This is done instead of cloning the DNA. In this chain reaction, small and short DNA strands (primers) are used which always compliment to the opposite ends of established sequence of a certain DNA strand that is being worked on (Kary Mullis, 2004)
Introduction
DNA technology is used whereby useful animals such as mice, guinea pigs are used because of the easiness in handling their DNA strand. Biotechnology and DNA engineering have given rise to molecular biology whereby scientists learn about genes and their biochemical composition. This has lead to development of new genetically modified organisms that allow scientists to venture in the world of DNA technology, invention and bio-analysis of biological materials whereby genetic engineering is in use. According to Kay-Ewe Wagner, beside continuous use of D.melanongaster, scientists prefer using mice because they are more close to human beings according to the diseases they suffer from. Mice are needed for studying genetic ailments like breast malignant tumor.
- Transgenic mouse. As the word Trans sound, transgenic is transferring of genes from foreign organism to another. Therefore, transgenic mouse can be defined as a mouse whose cells have been injected with a foreign DNA gene. This is done artificially in the laboratory. Two types of technique are applied in producing transgenic mouse. There is microinjection of genes in the zygote pronuclear and the other technique is by injecting the stem cells of the embryo into blastocoels.
- Knockout Mouse. As the word knockout (do away with) sounds, knockout mouse is a mouse whose biological functions have been interfered with artificially. This is achieved through deleting, mutating or erasing some vital genes or making them non functional in the DNA strand of the mouse. Knockout is one method of telling the function of a sequenced gene whose function is fully known. This can be achieved by deactivating a specific gene and studying the physical consequences.
- Advantages of Transgenic mouse. The use of transgenic mouse is simpler since the female and male pronuclear can be observed under light microscope as opposed to knockout technique. Therefore, the procedure can be controlled before fusing the two pronuclei into a zygote. Another advantage is that the derived embryo stem cells can be developed into any type of tissue, cell that needs to be studied. Going on, the embryo cells are used for modification of the mouse genome. This simplifies the work of DNA sequencing. This is because it is simple top insert, inject or remove the gene being dealt with. Lastly, it is through this method that knockout mouse is produced.
- Disadvantages of Transgenic mouse. It can be confusing and hard to identify the specific function of a certain gene. This is because in the process of transferring the genome, it can be interfered with. Therefore, it is not accurate.
- Advantages of knockout mouse. This method is very accurate since the scientists can probe quite easily the function of a specific genome. According to Marrio R., this can be achieved by deactivating a specific gene ion a mouse and then study the physical consequences of the gene in the same mouse. Knocking out the function of a gene gives reliable information as to what the gene does. Again, by studying the traits of the knockout mouse, researchers can use the same analogy to understand how the same gene can do in human beings or how it leads to human infection. Practitioners and researchers are using the same technology to investigate the molecular pathology of genetically diseases. This can help to discover the cure for such ailments.
- Disadvantages of knockout mouse. The main problem with this technique is that 16 percents of set up knockout mice are lethal. This is because genetically changed mice do not fully develop into maturity but they die before reaching the reliable stage of analysis. This calls for another expensive technique to be employed such as conditional mutations. Failure for the knockout mouse to develop into adults makes it difficult to tell and analyze the functions of specific genes and their effect to human diseases. Not all knocked genes give a positive results or identifiable change in the mouse. It can even give quite different traits from those seen in man if a similar gene is deactivated. For instance, mutation or transformation was observed in the p53 gene which is associated with human cancers. p53 leads to malignant tumors in a specific set of tissues. However, it is unbelievable that when the p53 gene is deleted out in mouse, the mouse gets malignant tumors in its tissues. Going on it is very hard to knockout some genes of genome loci. This is due to occurrence of repetitive genetic code sequence, extended DNA strand methylamine or multiple chromatin materials. According to Warner, mouse theories have different phenotypes different from human beings. This again, complicates this model.1
- Why or when, one technique is better than the other. Knockout mouse technique is a better method of telling the function of a sequenced gene whose function is fully known. This can be achieved by deactivating a specific gene and studying the physical consequences. Therefore, this technique is good to use when the specific function of a gene is known. On the other hand, if the modified organism need to be used for a long time it is good to apply transgenic technique that produces pluriotent cells that can be developed into any for scientific research.
Southern blotting technique
This is a technique employed by scientists to detect the specific location and presence of a particular DNA strand sequence in a DNA specimen. It is mostly used in molecular biology whereby there is combination of a gel of agarose type and electrophoresis method in order to separate various sizes of DNA strand. This is followed by transferring the size-separated DNA to a membrane that can filter it for probing in hybridization. This is according to Edwin Southern (1975).
Southern blotting includes use of restricted endonuclear which are used to cut DNA strands with high molecular weight into short fragments. These fragments are separated into size by use of agarose gel and electrophoresis method. The gel is treated with an acid if the fragments are bigger than 16kb before being blotted.
Polymerase chain reaction (PCR)
This is an artificial technique of duplicating or generating similar number of copies of a DNA strands into large numbers. This is done instead of cloning the DNA. In this chain reaction, small and short DNA strands (primers) are used which always compliment to the opposite ends of established sequence of a certain DNA strand that is being worked on.(Kary Mullis, 2005).
In this chain reaction model, shorter DNA primers are used. These primers must be compliment to the opposite ends of the DNA strand that is ready to be duplicated. In this technique, polymerization method is used to duplicate double stranded DNA from a template of a mono stranded DNA strand. One can use heat to unwide the double stranded DNA into two mono stranded DNA strand.
Typical example of producing a transgenic mouse using polymerase chain reaction
In this method of polymerase chain reaction, a stem cell must be obtained from a foreign organism but of the same species. In this case a white mouse and grey mouse was used to develop a transgenic mouse. In this experiment, the application of polymerase chain reaction was used in the integration of the marker gene or foreign gene (embryo stem cells).
- Procedure 1 The first step involved obtaining stem cells from white mouse blastocysts. This was followed by integrating the foreign gene from a white mouse to the genome of grey mouse through microinjection before duplicating genetic material of the grey mouse.
- Procedure 2 Then after integration, the resulted DNA gene sequence was cloned, screened and engineered. It was made sure that this sequence was almost similar to the original chain. After that, the new sequence was given a marker. (Polymerase Chain Reaction, M J McPherson and P Quirke- 1991)
- Procedure 3 Going on, the resulting stem cells from procedure 1 were combined with sequence from procedure 2 through DNA recombinant technique. This was done because the old and the new sequence were similar. After this step, antibiotics were obtained from step 1 to help in identification of the stem cells that did not incorporate the new strand.
- Procedure 4 The identified stem cells from procedure 3 were micro-inserted into the grey mouse embryo blastocysts. This was followed by implantation of the blastocysts (with the grey mouse stem cells and the white mouse with modified stem cells) in the uterus of the female mouse. Then the mouse was allowed to complete the pregnancy. The expected offspring should have character traits from the original ES and the modified embryonic stem cells. After the pregnancy period, the resulted baby mice had grey and white patches. Then the newly modified sequence was integrated into the germinal cells. This gave a crossbreed of pure white mice and partially white and grey newborn.
References
- Integrated DNA polymerase chain reaction and electrophoresis. Web.
- Jonathan P. (1994), Human polymerase chain reaction, Elsevier UK.
- Knockout and transgenic mouse. Web.
- McPherson AND Taylor G, (2003): Blotting and polymerase reaction, Academic Press, New York.
- Southern E, (1991): southern blotting and electrophoresis, Oxford University Press, New York.
- W hat is transgenic mouse. Web.
Footnotes
1 Wagner. The p. 53 knockout is a good example. p53 has been implicated in as many as half of all human cancers, but p. 53 knockout mice develop a different spectrum of tumors than do humans
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