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Southern Blotting By Erin Lucero Dr. Adema Bio 446 Who discovered Southern Blotting? • Sir Edwin Mellow Southern discovered Southern Blotting in 1975. • At the time of the discovery he was working at Medical Research Council Mammalian research unit at Edinburg • Founder, Chairman, and Chief scientific officer of Oxford gene technology. • Emeritus professor of biochemistry at the University of Oxford. • Along with discovering Southern Blotting, his company developed the DNA microarray. No Sequencing or PCR! PCR machine • Before Sequencing or PCR individuals relied on Southern blotting to detect genes through out the entire genome. • Can use Southern Blotting to detect genetic markers such as, Restriction Fragment Length Polymorphism. • Blotting pattern is characteristic to a specific genotype at a specific locus. • Can use this technique to detect diseases Next Generation Illumina Sequencing machine Methodology of Southern Blotting • 32 P Isotope • The exact sequence of the probe was unknown • The DNA probe was specific to a certain region of DNA, usually isolated from a specific chromosome. • Hybridization between complementary DNA and Probe. • Denature the ddDNA into ssDNA with PH, then load it into gel. Southern Blotting Edmund Southern graphical representation of Southern Blotting in 1975. The manuscript was rejected by Journal of Molecular Biology, E. Southern, "Detection of specific sequences among DNA fragments separated by gel-electrophoresis," J Mol Biol, 98:503, 1975. (Cited in 30,666 papers)” Restriction Fragment length polymorphisms (RFLP) • Polymorphism: is a genetic variant that appears in at least 1% of a population • Such variations include: ABO blood type, Rhesus factor, and major histocompatibility complex (MHC). • RFLP are differences in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA. • Moreover, RFLP is a sequence of DNA that has a restriction site on each end with a "target" sequence in between. A target sequence is any segment of DNA that bind to a probe by forming complementary base pair. • RFLP can be used to detect certain deleterious diseases, such as, sickle cell anemia, and cystic fibrosis. • The DNA is polymorphic among certain sites. • When a single nucleotide changes in gene it is denoted as Single Nucleotide polymorphisms. Clinical Aspects of RFLP • Genome mapping • Paternity testing • Genetic disorders • DNA fingerprinting RFLP Analysis References • https://en.wikipedia.org/wiki/Edwin_Southern • https://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism • https://employees.csbsju.edu/hjakubowski/classes/ch331/dna/NA_4C1_Lang_DNA.html • http://www.the-scientist.com/?articles.view/articleNo/15193/title/The-Birth-of-theSouthern-Blot--1975/ • http://www.biology-pages.info/R/RFLPs.html • https://www.britannica.com/science/polymorphism-biology • ttp://www.chem.fsu.edu/chemlab/bch4053l/Medical%20Biochem/sequence/backgroun d.html • http://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism(RFLP)-Technique.aspx