MBIO 3410 Lecture Notes - Lecture 3: Frederick Sanger, Ion Semiconductor Sequencing, Pyrosequencing
24 Dec 2015
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Document Summary
Collection of fragments whose length depends upon the position of a particular base: i. e. when by chance a ddntp was incorporated, ddntp are dead ends ; no dntps can be added as there is no. Four reactions and four sets of fragments. Resolved on a long denaturing page gel: add some alpha 32p dntp, need denaturing polyacrylamide gels. Automated and four reactions can be combined as 4 different colours can be used. Parallel sequencing: millions of reactions performed at the same time (automation) Illumina (solexa) sequencing: time, accuracy, and cost. Sequence determined as dna polymerase adds nucleotides. Pyrophosphate released during formation of phosphodiester bond. Pyrophosphate used to make atp: used by luciferase to release light, apyrase degrades all dntps, now next dntp can be added to challenge the dna polymerase. Principle: challenge dna polymerase with one nucleotide at a time: if incorporated light signal, if not incorporated no signal. One bead per well plus packing beads (contain bound enzymes)
