Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber’s hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. It can also be used in the identification of the causative agent of any infectious disease.Mitochondrial disorders are clinically and genetically heterogeneous.It is used in DNA fingerprinting and restriction fragment length polymorphism.Used for paternity testing along with the identification of criminals and victims.The technique can be used for DNA analysis to detect point mutations and other structural rearrangements in the DNA sequences.It can be used in diagnostic study of genetic diseases.It is applied in the field of gene mapping and evolution.It is used to detect and determine the size of specific DNA fragment in the given mixture of genomes.If a fluorescent or a chromogenic dye is used, the hybridized regions can be visualized on X-ray film or as colored spots on the membrane.The hybridized fragments on the membrane can be detected by autoradiography.Detection and visualization by autoradiography: The base pairing between the probe and ssDNA on the membrane forms a hybrid DNA molecule by the process called hybridization.ħ.The probe can be labeled with any radioactive substance or dyes with fluorescence or chromogenic property.The membrane is then incubated with a labeled probe which contains the complementary sequences to the gene of interest on the membrane.The size of the DNA fragments can be determined by comparing their relative size with the DNA bands of known lengths.The position of the band containing these hybridized fragments is determined by autoradiography or any other method depending on the types of probes used.The probe hybridizes with the immobilized ssDNA on the membrane having the gene of interest.The membrane is then incubated with an appropriate radiolabeled probe (radiolabeled ss DNA or radiolabeled RNA) specific for the gene of interest.After transfer, the strands on the membrane surface are immobilized by baking or UV-radiation.The resulting single stranded DNA is then transferred onto a nitrocellulose or nylon membrane either by capillary blotting or by electroblotting.The gel is then placed in alkaline salt solution to denature the double stranded DNA.In Southern blotting, DNA is first cut with restriction enzymes and the fragments are separated according to size by agarose gel electrophoresis.This process of base pairing between complementary single-stranded DNA molecules is known as hybridization.This technique is based on the formation of hybrid DNA molecules when homologous, denatured DNAs from two different sources are mixed with each other under the appropriate conditions of ionic strength and temperature.You may also want to see: Western blotting: Principle, Steps involved and Applications This technique helps in the identification of a particular DNA fragment containing the gene of interest.This technique is named after its inventor Edward N.Southern blotting also called Southern hybridization is a hybridization technique which helps to detect specific gene sequences or DNA fragments within complicated mixture of nucleic acids.
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