MBIO 4440 Lecture Notes - Lecture 10: Sanger Sequencing, Massive Parallel Sequencing, Capillary Electrophoresis
13 Feb 2019
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Document Summary
Sanger sequencing is a robust method and after three decades of improvements it can achieve read-lenghts of up to 1,000 bp and per-base accuracies as high as 99. 999%. An important improvement was the use of capillary electrophoresis to separate the fluorescent truncated dna molecules synthesized during the sequencing reaction. The method was scalable to high-throughput approaches with sequencing costs as low as. Yet, the chemistry of the reaction, and the type of platform involved prevented its continued development for a more cost-effective approach. Bottlenecks of sanger sequencing: sample preparation: dna has to be randomly fragmented and cloned into plasmids, which in turn are used to transform e. coli. Hundreds of thousand samples need to be arranged into 96 or 384 format. This involves lots of pipetting, use of consumables etc. Genomic libraries are very time consuming and use lots of preparation.
