PCR and Southern Blotting: Applications in Medicine

PCR and Southern Blotting Applications in MedicineIntroductionThe 1970s introduced an inspirational technique in which a specific sequence could be determined from a render of desoxyribonucleic acid via the process of Southern blotting. This method, named after Edwin Southern, provided the basis for a range of common laboratory techniques such as western blotting, eastern blotting and northern blotting 1.Shortly after, in 1983, a revolutionary method was invented by Kary Mullis, called the polymerase chain reaction (PCR) 2. This procedure was originally utilize to amplify and aim deoxyribonucleic acid sequences in the human genome. Its use in hereditary digest was immediately recognised, as one of the first publications of its use was of prenatal diagnosing of sickle-cell anaemia 3. Since then it has been manipulated and a whole array of techniques have been derived from this invention. both(prenominal) PCR and Southern blotting have been employ astray in understanding and identifying microbes which in turn assists the diagnosing and management of patients suffering from infectious diseases.PCR ProcedureThe sample of deoxyribonucleic acid is heated up to 90C to separate the devil strands of DNA thereby exposing the nucleotide reports on each strand. A undercoat is then annealed to each strand from the 5 region at round 60C and the temperature is increased once more than. The thermostable DNA polymerase enzyme, Taq, prevents contamination by binding free completing base pairs to the original strand of DNA at temperatures up to 70C. The strands ar then cooled and double the measure of DNA is synthesised, and the cycle restarts until a sufficient amount of DNA is produced.Reverse-transcriptase PCR (RT-PCR) is employ when the original sample of RNA is transcribed so that DNA is the proceeds of amplification. The sensitivity of PCR is great, as espial is from a single nucleotide base whilst its quantitative ability is derived from the proportio nal expansion of amplified DNA from its original size of it 5.Southern Blot ProcedureSouthern blotting begins 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 undergo agarose gel ionophoresis where the fragments diffuse across a polarised field according to their size. The DNA is denature by sodium hydroxide and transferred to a sheet of nitrocellulose or nylon and incubated with a hybridisation probe of single-stranded DNA. This radiolabelled probe binds to the exposed complementary base pairs and can be detected by autoradiography 6.Southern Blotting Applications in Medical MicrobiologySouthern blotting is primarily use for DNA fingerprinting, gene sequencing and genetic engineering.It has been use in the identification of strains in microbes such as last the type of human papillomavirus extracted from a condyloma. However in this case it provided to be unreliable as it produced fa lse-negatives, as PCR and in-situ hybridisation were deemed to be more efficient 7. Another use of Southern blotting was in the detection of a strain of Listeria monocytogenes. In this study it was deemed an important technique in support species identification and in the characterisation of epidemic strains 8.This method can be used to DNA fingerprint most microbes and determine a diagnosis and treatment for a patient suffering from their pathogenicity. However it is found to be too grievous, time consuming and requires large amounts of high quality DNA for most routine laboratories yet variations of this technique are still widely performed. The western blot, which uses antibodies as the probe to detect proteins instead of DNA, is a substantiating test in the diagnosis of a human immune-deficiency virus(human immunodeficiency virus) infection 9. Since the naturalised use of PCR there has become a decreased need for these laborious techniques in DNA sequencing 10.PCR Applicati ons in Medical MicrobiologyPCR can be used in detecting the genetic sequence of all microbes. It is useful in detecting organisms in early cultures where organisms are otherwise difficult to isolate, for good example in enteroviruses RT-PCR is more sensitive than culture and the gold standard is detection of the this genome in cerebral spinal fluid (CSF) by PCR 11.PCR is also used in detecting genes encoding antibiotic resistance such as in Helicobacter pylori and Methicillin-resistant Staphylococcus aureus. However its use is currently unsuitable for the diagnosis of H. pylori as clinical samples may contain inhibitors which can generate false-negatives10.PCR is used in quantifying the viral load of HIV within an infected someone then determining the effectiveness of their treatment. The problem occurs when the genome sequence of the HIV changes therefore the PCR method needs to be altered and the current test would found useless9. Currently immunoassays are used in the diagnos is of a HIV infection, however early in infection there is a varying period of time until anti-HIV antibodies can be detected and this provides a effectiveness area of identification of the HIV by PCR 12.Pitfalls of PCR include that the sample must be kept cold during storage and transport to the laboratory, the expertise needed for analysing and interpreting results as well as standardising between different laboratories and eventually the expensive cost of the procedure compared to available techniques9.ConclusionThe applications for PCR and Southern blotting are enormous however they have limitations which prevent it from existence routinely used in the diagnostic laboratory. With the advance of technology both are being developed, especially PCR, and have endless applications in medical microbiology.