Cycle sequencing is a simple method in which successive rounds of denaturation annealing and extension in a thermal cycler result in linear amplification of extension products From applied ID: 1045577
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1. 1980s: Linear PCR is used to generate more products from less templateCycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products. From applied biosystems.com
2. Current Sanger sequencing use dideoxy terminators that have different fluorescent dyes attached Dyes are attached to the ddNTPsOnly one reaction tube per sample, instead of four (isotope method)Fluorescent fragments are generated by incorporation of dye-labeled ddNTPs. Each different dideoxy nucleotide (ddATP, ddCTP, ddGTP, or ddTTP) carries a different colored dye. All terminated fragments (those ending with a ddNTP) contain a dye at their 3’ end Only one lane/sample on gel, instead of four (isotope method)From Applied Biosystems
3. Overview of procedureTube contains all four fluorescently labelled dideoxynucleotidesLinear PCR is used to generate multiple rounds of terminated fragmentsSample is loaded in one capillary
4. The output of the detector can be sent directly to a computer and the DNA sequence recorded (in real time) as the gel is being run…Example of output: