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DNA Sequencing

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Published in: Biology
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DNA Sequencing - A Simple Explanation. 

Soni / Faridabad

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  1. DNA SEQUENCING DNA sequencing is a biological method for determining the order of the nucleotide bases, adenine, guanine, thymine, cytosine in a DNA sequence. Different type of methods used for DNA sequencing, Chain termination method Chemical degradation method Pyrosequencing method CHAIN TERMINATION METHOD IN 1977, feredrick Sanger and his co-worker developed chain termination method. In this method derivative of deoxyribonucleoside triphosphate is used i.e. dedeoxyribonucleoside triphosphate that lack a 3'OH group. Purified DNA is synthesized in vitro in a mixture that contains single- stranded molecules of DNA which act as a template for DNA sequencing. DNA polymerase enzyme and four bases dATP, dGTP, dCTP, dTTP are started the reaction. Dideoxyribonucleotide are also present in a nucleotide mixture, it can incorporated into a growing DNA chain. Because this chain now lack 3'OH group, addition of next nucleotide block and DNA synthesis stop. To determine the complete sequence of DNA fragment, the double stranded DNA is first separated, act as a template for sequencing. Four different chain termination dideoxyribonucleotide triphosphate are used in four separate DNA synthesis reactions on copies of the same single stranded DNA template. Each reaction produces a set of DNA copies that terminate at different points in the sequence. The products of these four reactions are separated by electrophoresis in four parallel lanes of a polyacrylamide sequencing gel. Sequencing gels are run in the presence of denaturing agent Urea. The function of urea is to prevent DNA secondary structure due to intrachain base pairing, which affect electrophoretic mobility.
  2. p p OCH2 o Dideoxynucleotide (ddNTP) p p OCH2 o Base Base Deoxynucleotide (dNTP) STRUCTURE OF dNTP and ddNTP The newly synthesised fragments are detected by a label either radioactive or fluorescent that have been incorporated either into prime or into one of the deoxyribonucleoside triphosphate used to extend the DNA chain. In each lane, the bands represent fragments that have terminated at a given nucleotide, but at different position in the DNA. By reading of the band in order, starting at the bottom of the gel and working across all lanes, the DNA sequence of the newly synthesised strand can be determined.

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