Posted at 10.13.2018
The 1970's created an inspirational strategy when a specific sequence could be driven from a sample of DNA via the procedure of Southern blotting. This technique, called after Edwin Southern, provided the basis for a range of common lab techniques such as western blotting, eastern blotting and northern blotting .
Shortly after, in 1983, a groundbreaking method was developed by Kary Mullis, called the polymerase chain response (PCR) . This process was actually used to amplify and identify DNA sequences in the individual genome. Its use in genetic evaluation was immediately accepted, as one of the first magazines of its use was of prenatal examination of sickle-cell anaemia . Since then it's been manipulated and a complete array of techniques have been produced from this invention.
Both PCR and Southern blotting have been used extensively in understanding and discovering microbes which assists the analysis and management of patients suffering from infectious diseases.
The test of DNA is heated up up to 90C to separate both strands of DNA thus revealing the nucleotide bases on each strand. A primer is then annealed to each strand from the 5' region at around 60C and the temperatures is increased once again. The thermostable DNA polymerase enzyme, Taq, helps prevent contaminants by binding free complementary platform pairs to the initial strand of DNA at conditions up to 70C. The strands are then cooled and twice the quantity of DNA is synthesised, and the circuit restarts until enough DNA is produced.
Reverse-transcriptase PCR (RT-PCR) is used when the original test of RNA is transcribed so that DNA is the merchandise of amplification. The sensitivity of PCR is fantastic, as recognition is from an individual nucleotide platform whilst its quantitative ability is derived from the proportional growth of amplified DNA from its original size .
Southern blotting commences with a sample of DNA which is first broken up by a restriction endonuclease into smaller, varying fragments. The DNA is then placed into wells to endure agarose gel electrophoresis where the fragments diffuse across a polarised field matching with their size. The DNA is denatured by sodium hydroxide and used in a sheet of nitrocellulose or nylon and incubated with a hybridisation probe of single-stranded DNA. This radiolabelled probe binds to the revealed complementary basic pairs and can be discovered by autoradiography .
Southern blotting is mainly used for DNA fingerprinting, gene sequencing and hereditary engineering.
It has been used in the recognition of strains in microbes such as concluding the type of human papillomavirus extracted from a condyloma. Yet, in this case it provided to be unreliable as it produced false-negatives, as PCR and in-situ hybridisation were regarded to be more effective . Another use of Southern blotting was in the detection of the tension of Listeria monocytogenes. Within this study it was regarded an important strategy in verifying kinds id and in the characterisation of epidemic strains .
This method may be used to DNA fingerprint most microbes and determine a examination and treatment for a patient suffering from their pathogenicity. Nonetheless it is found to be too laborious, time consuming and requires large amounts of high quality DNA for most tedious laboratories yet variations of this strategy are still broadly performed. The american blot, which uses antibodies as the probe to identify proteins rather than DNA, is a confirmatory test in the examination of a individuals immune-deficiency virus(HIV) contamination . Since the proven use of PCR there has become a reduced dependence on these laborious techniques in DNA sequencing .
PCR can be used in detecting the genetic sequence of most microbes. It is useful in detecting organisms in early civilizations where organisms are normally difficult to isolate, for example in enteroviruses RT-PCR is more very sensitive than culture and the 'gold standard' is recognition of the this genome in cerebral spinal fluid (CSF) by PCR .
PCR is also found in detecting genes encoding antibiotic amount of resistance such as in Helicobacter pylori and Methicillin-resistant Staphylococcus aureus. However its use is currently unsuitable for the identification of H. pylori as scientific samples may contain inhibitors which can make false-negatives10.
PCR is employed in quantifying the viral weight of HIV within an infected person therefore identifying the effectiveness of their treatment. The issue occurs when the genome sequence of the HIV changes therefore the PCR method needs to be changed and the existing test would verify useless9. Currently immunoassays are being used in the diagnosis of a HIV an infection, however early in infection there is a variable time frame until anti-HIV antibodies can be recognized and this provides a potential area of id of the HIV by PCR .
Pitfalls of PCR include that the test must be retained cold during storage area and carry to the laboratory, the expertise necessary for analysing and interpreting results as well as standardising between different laboratories and finally the expensive cost of the task in comparison to available techniques9.
The applications for PCR and Southern blotting are substantial however they have constraints which prevent it from being regularly found in the diagnostic lab. With the advance of technology both are being developed, especially PCR, and have countless applications in medical microbiology.