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The study of sequences of genetic material in any organism is important in helping scientists analyze the whole of the organisms genome. This way, causes of genetic disorders can be easily identified and therefore addressed. One of the most commonly used method in studying DNA sequences is Nucleic acid hybridization due to its efficiency (Lee et al., 1991). It does not only help in identifying gene sequence and function, but also helps in identifying regulatory measures of such genes (B.Noonberg, 1995). This experiment is aimed at understanding the real mechanism involved in genetic material sequencing through the nucleic acid hybridization.
The basis of this technique is the ability to join two strands together. It entails the use of a probe which, with the help of its fragments of DNA or RNA, detects complementary strands from a mixture as the plasmid used gets digested by a restriction enzyme. The obtained fragments are used in agarose gel electrophoresis where they get separated into single strands which are then transferred to membranes that are capable of binding DNA. A single stranded probe is then added onto which the desired sequence binds and later labeled using markers such as radioactive molecules or molecular markers (Hennig, 1979, Tsao et al., 1983).
In this case, two plasmids were used pMAQ105 and pACYC184 with the aim of identifying the aadB gene. A 37 kb cloning vector, pACYC184 was used which consisted of two plasmids, R388 and pMAQ28. The required aadB gene is 59 bp long and is contained in the pMAQ28 plasmid. The pACYC184 plasmid acts as the probe since it consists of the aadB which binds to the homologous plasmid. pMAQ105 plasmid consists of the aadB gene as well as other genes and restriction sites (Holmes et al., 2003).
The pMAQ105 probe is labeled using the fluorescent technique which involves attaching a fluorophore onto to the probe. The labeled probe can then later be detected by addition of an alkaline antigen conjugate whose phosphate enzymes reacts with the fluorescent. CPD is then added to detect the phosphates in the labeled molecule containing the desired DNA sequence.
The experiment was started by adding the restriction enzymes obtained from set 2 above onto three samples to prepare the fragment of DNA from the pACYC184 plasmid. The samples were then incubated and later added on agarose gel during which they resulted into DNA fragments of different sizes.
The gel containing the DNA fragments was used for southern blotting where the gel was added HCl. The solution was then stricken lightly and the gel was cleansed through addition of distilled water. The gel was soaked in 0.4m of NaOH after which a nylon membrane, a zeta probe, was placed on top of the gel then a whatman paper followed. A paper towel that had been prepared previously alongside the nylon membrane and the whatman paper was then placed after the whatman paper together with a glass plate weighing 500g.
Since the pMAQ105 plasmid contains the EcoRI restriction site, the EcoRI digestive enzyme was added to the pMAq105 plasmid after which the solution was incubated for about two hours. The nylon paper which was used as a zeta probe in the southern blotting technique was later obtained and was then soaked in a SSC buffer. The nylon membrane was then placed in a hybridization bag where a DNA carrier alongside a pre-hybridization buffer was also added. The hybridization bag was then tightly closed to prevent the formation of air bubbles and was then incubated for a period of two hours.
The hybridization buffer and the carrier DNA were added to a labeled probe which was provided. The probe was then boiled to denature it and then added on ice. After denaturing, the probe was then added to the hybridization buffer after it had been equilibrated at 60°C. The bag that had been closed containing the DNA membrane was the opened and the previously added pre-hybridization buffer discarded and the probe DNA mix added to replace it. The sample was then hybridized for 12 hours at temperatures of 600C.
The membrane was later obtained from the bag and placed in a weighing tray where the SSC buffer was added together with SDS and the sample solution incubated for a few minutes. The solution from the sample was discarded and the process repeated. The membrane that was obtained was covered using buffer 1 after which it was recovered again and covered with buffer 2. The membrane was then added onto an antibody conjugate and incubated with regular gentle agitations. It was then washed using the previous buffer 1 followed by buffer 3.
After this, the membrane was placed on another weigh tray where CDP-star was added and the solution incubated for about five minutes. Tissue blotting paper was used to get rid of the excess solution. A plastic wrap was used to wrap the membrane which was then photographed and compared with another from the auto radio graph. The region of the pACYC184 plasmid that was homologous to pMAQ105 was determined.
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