Health Molecular Biology Techniques

Question: Discuss about theHealthfor Molecular Biology Techniques. Answer: Introduction The use of molecular biology techniques has been widely adopted since the inception of DNA technology. Therefore scientists have been using various methods employing the DNA and the genes to provide solutions to the pharmaceutical industry and healthcare needs. The peptide nucleic acid method is preferred because it is less time consuming than the oligonucleotide techniques. However, the peptide nucleic acid method requires the use of laboratory reagents that are very expensive. Some factors which affect the success of in situ hybridization are the temperatures used, the length of time taken and the concentration of the reagents. This paper explores a research carried out to make an in situ hybridization protocol and make a comparison between the oligonucleotide probes and use of peptide nucleic acid probes methods. Optimizing the Temperatures Under normal circumstances, the in situ hybridization is good when carried out at room temperatures. In this article, various temperatures such as 37, 55 and 65? were explored. At the 37, the slides containing the tissues stained well indicating brown signals which were more than the experiment carried out at room temperature. At 55 ? the signals were more than the 37 ?but the background was not clear, while at 65 ?, stained poorly and the brown signal was not clear enough while the background was poor. The variations I the clarity of the brown signal and the background indicate that the temperatures in oligonucleotide hybridization could be optimized. Therefore it was suggested that the optimal temperatures were between 37 and 55 ? and hence the average of the range was taken as 45 ?. Incubation Time for Proteinase K The proteinase K is used for maintaining the morphology of cells and aids in the tissue digestion so that the access by the hybridization probes as well as the linker antibodies can be achieved. Under standard conditions, the incubation period for proteinase K should be thirty minutes at room temperature. In this case, the ten, fifteen and twenty minutes incubation periods were investigated. Both time ranges were able to produce brow signals but at different intensities. The twenty minutes incubation produced a browner signal than the fifteen minutes which in turn had a stronger brown signal than the ten minutes incubation. However, the thirty minutes period had the strongest brown signal but above thirty minutes can be time-consuming and cause the rapture of the cells being investigated. Optimized Temperatures at Different Periods Time The time allowed for the whole process of in situ hybridization is very crucial. This because there is an existing evidence that specific binding actually requires more time than a nonspecific binding of the reactant in order to attain a state of equilibrium. This would then mean that a longer period of hybridization is mostly preferred. However, the hybridization time mostly depends on several factors such as the methods of detection, accessibility of the DNA by the probes, the length of the probe as well as the rate of the diffusion of the coefficients. The standard in situ hybridization protocols requires a hybridization period of two hours at room temperature. In this article, one hour and two hours were investigated but at different temperature intervals of room temperature, 37, 45 and 55 ?. At a period of one hour at room temperature and 37, the signals were very weak. However, at 45 and 55, the signals were strong as compared to the room temperatures and 37 for the same period of time. This means that it is possible to get positive in situ hybridization at low time intervals. When the time was increased to two hours, the intensity of the signal was higher as compared to the one-hour time frame. However in this case, at 45 ?, more intense brown signals and background was observed as compared to the room temperature, 37 and 55 ?. This is logical because in the temperature optimization, a temperature range of 37 to 55 ? had been considered and an average of 45 ? had been arrived at. The investigator performed the in situ hybridization for different lengths of time such as half an hour, one hour, one and a half hours and two hours to find out whether the time could be reduced. It was found out that for half an hour at 45 ? and for one and a half hours at 55 ?, similar in situ hybridization results as in the two hours at 45 were obtained. It was noted that for half an hour at 45 or 55 ?, the hybridization time was reduced with a production of strong brown signals. Thus when the time period is reduced, the temperatures can be increased so as to get strong signa ls in oligonucleotide in situ hybridization. Use of Water In standard methods, the diethylprocarbonate solution is used as an alkylating agent to inactivate any proteins which could be present in the reactants. In this case, tap water and distilled water were tested. The tap water indicated a weak signal and a lot of background staining while the distilled water showed a strong signal with a little background staining. This means that even distilled water can be used in place of diethylpyrocarbonate during oligonucleotide in situ hybridization. The Concentration Incubation Period of Stringent Wash Buffer The stringent wash buffer is used to prevent the possibility of nonspecific binding of the oligonucleotide probes. The standard concentration is 2** for thirty minutes. In this optimization, a concentration of 4** was used for varied periods of time which were, ten, fifteen twenty and thirty minutes. The incubation period of ten minutes gave bad results while the fifteen, twenty and thirty minutes gave similar results with a lot of background staining. Therefore a high concentration leads to background staining while a short period causes nonspecific staining. This means that a 2** concentration for thirty minutes is still the preferred condition. Peptide Nucleic Acid Probes The use of peptide nucleic acid probes is preferred because they are modified probes which mimic the DNA. Their modification enhances the binding capacity as opposed to the oligonucleotide. Several tissues such as the thymus, liver and bone tissues investigated to determine the binding capacities of the two probes. In the liver tissue, both probes had a good staining although the peptide nucleic acid probe had a cleaner staining. In the bone tissue, the oligonucleotide probe had a negative result while the peptide nucleic acid probe had a positive staining. The thymus tissue had a positive staining when both probes were used although the peptide nucleic acid had a stronger staining making the microscopic examination hard. It was concluded that the peptide nucleic acid Peptide nucleic acid probe is more preferable because of its intense staining properties as well as the short time taken. However, with the optimization of the in situ protocols, the oligonucleotide probe can be used to achieve the same efficiency as the peptide nucleic acid probes. Conclusion The optimization reports that incubation period is efficient when reduced from two hours to thirty minutes. The distilled water can be used instead of the diethylprocarbonate solution. The temperature ranges were found to be between 37 and 55 ? while the oligonucleotide probes can be used due with the optimized conditions.