Sanger Sequencing

Text from Jenny Draper's Doctoral Thesis. Please change. — Jenny Draper 2010/03/30 16:14

DNA sequencing was initially performed using 32P- radiolabeled DNA either chemically fragmented at specific base pairs (Maxam-Gilbert sequencing¹⁾) or chain-terminated at specific nucleotides (Sanger sequencing²⁾). By simultaneously separating each population of fragments on a gel, the sequence could be “read” as a band in one of the four (A|T|G|C) lanes. Utilizing his dideoxy chain-termination method, Fred Sanger’s team completed the first genome of a DNA-based organism, the 5,386 base pair bacteriophage FX174, in 1977³⁾. It wasn’t until 1986, however, when Leroy Hood and colleagues modified the classic Sanger sequencing technique to include differently colored fluorescent dyes for each of the four sequencing reactions, that DNA sequencing became automatable ⁴⁾. The entire sample could be loaded at once, and a scanner could read the color of each band as it passed by, thus recording the sequence. This is still the most commonly used method of DNA sequencing, and implementation of the technique has improved greatly with time. The UC Berkeley DNA Sequencing Facility, which many UCSC labs use to sequence their DNA PCR products, currently uses two Applied Biosystems 3730xl DNA Analyzers, which are capable of running 4,224 samples per day with up to a 500 bp resolution, or 768 samples per day with up to a 900 bp resolution⁵⁾.