The Development of Reverse Transcriptase Real Time PCR Methodologies - Coursework Example

Real time PCR Polymerase chain reaction (PCR) allows the short DNA sequences to be amplified exponentially within a DNA molecule that is longer and double stranded. In PCR, a pair of primers is used whose length is about 20 nucleotides, which are complimentary to a sequence that is defined on the two DNA strands. For a copy to be made of the sequence that is designated, DNA polymerase is used to extend these primers. One of the reasons that made reverse transcriptase-PCR non-quantitative is the fact that ethidium bromide is a stain that is rather insensitive. Competitive PCR was a method that was designed to try and make the whole method a little bit more quantitative, but it has proved to be very time-consuming as well as cumbersome. The development of reverse transcriptase real-time PCR helped to address some of these problems (Jan Schrader, et. al., 2003). It allows a DNA molecule to be copied partly using DNA enzyme polymerase, which is tolerant to temperatures levels that are elevated. Reverse transcriptase allows mRNA to be copied to cDNA using a primer dT oligo. Random oligomers can be used in some instances. Reverse transcriptase, which possesses an endo H activity, is often used in real-time PCR. One the main reason why this is desirable is so that it can allow for the formation of a second strand of DNA by removing the mRNA. A mix of PCR is then set up, and this includes; a suitable buffer, specific primers, heat-stable polymerase and deoxynucleotides. It is worth noting that the development of PCR was informed by various reasons. First, there was a need for differences to be quantitated in mRNA expression. Secondly, the fact that mRNA is only available in small amounts in some procedures also led to its development. Such procedures include the use of primary cells, precious reagents and tissues which are small in amount. For quantitation of mRNA to be achieved, various methods are available. They include RPA, northern blotting as well as PCR. The most sensitive among these is PCR, and it can also discriminate mRNAs that are closely related (Jan Schrader, et. al., 2003). There is no amplification that is involved in RPAs and northern blotting. Northern blotting is a technique that experts think should be embraced because it is very efficient, although the amount of RNA that it requires is in most cases more than can be availed. If it has been established that RNA amounts are low, then the PCR methods will be very valuable. As opposed to analysis by agarose, the results given by reverse transcriptase real-time PCR are quantitative. Another advantage that it has is the relative ease with which it can be used as well as its convenience. This is in stark contrast to some of the methods that were in use before. For loading control purposes, the gene to be used should have the following features. First, all cells of the gene should have equal copy numbers. The gene should also be expressed on all the cells that are available. For purposes of correction which should be accurate, medium copy number is desirable. Among the standards that are most commonly used include the following; Beta actin mRNA, 28s and MHC mRNA. With each cycle of PCR, the amount of DNA doubles (Jan Schrader, et. al., 2003). This method offers various advantages such as ensuring that the probability of contamination, as well as variation, is reduced significantly. This technology has also helped to make online monitoring easy. Signals, which are fluorescent, are observed keenly during their generation after rising above the background, but just before reaching the plateau. For real-time PCR, various fluorescent probes can be used. Some assays favor the general dyes, as opposed to DNA that is double stranded. This is because they bind preferentially. Others favor reagents that are sequence-specific and an example of this is exonuclease probes, molecular beacons and hybridization probes. Sequence-specific probes are more expensive, but the specificity that they add to the assay cannot be ignored. They also enable multiplexing applications. It can be observed that the dynamic range of real time PCR is much greater than that of the other techniques. There is the elimination of post-reaction processing. The exponential range is used to take the measurements, where the concentrations of the component are not limiting. It also allows for the automation of the process as a whole thus giving it a great advantage. This technique uses reporter molecules that are fluorescent to observe how amplification products are produced during every phase of the reaction. This leads to the combination of the detection steps and the nucleic acid into a single homogeneous assay. This ensures that the detection of amplification products by the gel electrophoresis is obviated. When the appropriate data analysis and chemistries are used, then the need for DNA sequencing and southern blotting is eliminated. The fact that this technique is simple, sensitive all coupled with its specificity sets it apart from the other techniques (Jan Schrader, et. al., 2003). Improved protocols, reliable instrumentation and the fact that new chemistries have been introduced make reverse transcriptase RT-PCR a technology that cannot be assumed if the comparison and detection of different RNA levels is to be done efficiently and successfully. The reverse transcriptase real time PCR has helped in the diagnosis control of renal cell carcinoma. This has been found to originate from the proximal convoluted tubules. It is a form of cancer that affects the kidney. The fact that the technique has led to automation of tasks has helped make monitoring of patients easier. Different stages in predicting the prognosis of the condition have now been made easier. They include radiology, staging, histopathology as well as cryoablation. In the metastatic stage of this condition, the disease spreads to the organs of the body. The reverse transcriptase RT-PCR would come in handy in ensuring early detection through the consistent monitoring of the patient’s body. Renal cell carcinoma is ranked as one of the most common types of malignant renal tumors. It is a kind of kidney cancer that experts say grows about 1 cm a year in diameter. It is not easy to detect it, and that is why the methodologies of reverse transcriptase RT-PCR were very important. It has aided in controlling this kind of kidney cancer by ensuring that it does not grow and spread as aggressively as before. Reference list Jan Schrader, et. al. 2003. Application of Real-Time Reverse Transcriptase-Polymerase Chain Reaction in Urological Oncology. The Journal of Urology Volume 169, Issue 5 , 1858- 1864. Read More