DNA SEQUENCING.. A VERY LONG STORY!!!
Hello all, I am here to present an important test which gained importance recently viz. DNA Sequencing Procedures followed: Maxam and Gilbert Method & Sangers method. Basic principle involved in either method: Series of DNA fragments are generated with a common starting point but with variable termini. Illustration by Sangers method Firstly in order to determine the base sequence in the segment of DNA, large number of identical copies of the DNA fragment must be obtained. Well, the sequencing molecule may be too small such as viral genome or a plasmid or in some cases, the sequence may be a part of large molecule. Sangers method uses single stranded DNA. Hence forth the dsDNA must be digested with a nuclease enzyme or cloned in a ss vector (Eg: Bacteriophage M13). The real steps begin now... Sequencing of the ss DNA fragments is carried out by generating a series of nested fragments by incubating the fragment with DNA Polymerase, a short primer, four deoxy nucleoside triphosphates and a small amount of dideoxy nucleoside triphosphate. Let us see how this work.. ss Base sequence of DNA: ---------ACGAATACGATCCATGCG ---------T PRIMER Mix1: dATP, dCTP, dGTP, dTTP, ddATP This mixture will terminate polymerization reaction at position T Just like this.. -------TGCTTddA -------TGCTTATGCTddA -------TGCTTATGCTAGGTddA Mix2: dATP, dCTP, dGTP, dTTP, ddCTP This mixture will terminate polymerization reaction at position G Just like this.. -------TGddC -------TGCTTATGddC -------TGCTTATGCTAGGddC --------TGCTTATGCTAGGTACGddC Mix3: dATP, dCTP, dGTP, dTTP, ddGTP -------TddG -------TGCTTATddG -------TGCTTATGCTAddG etc Mix4: dATP, dCTP, dGTP, dTTP, ddTTP Chance incorporation of dideoxy thymidine which lacks 3' hydroxyl group terminates polymerization at that point. -------TGCddT -------TGCTddT -------TGCTTAddT -------TGCTTATGCddT -------TGCTTATGCTAGGddT I tried my level best in giving you a glimpse of this sequencing and I hope I could meet your anticipation. If you think its time to clap..then you are definitely amiss. Post interval part goes like this.. After successful incubation, the mixture now contains radioactive DNA strands with variable termini. The mixture is then denatured and electrophoresed to separate the newly synthesized strands based on size. The position of the radioactive band is determined by X-ray development. DNA sequencing is primarily used in genetic problems, to determine the organismic relationships. Ref: Chapter 13: The Classification of Phylogeny of Bacteria,Principles of Fermentation technology By Peter F Stanbury,Allan Whittaker,Stephen J Hall 2nd Edition Elsivier Publication, Page no: 321-322. THIS BLOG DOES NOT CONTAIN ANY PLAGIARIZED MATERIAL.