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Genomics 101: an introduction to sequencing

Published on May 30, 2021   28 min

A selection of talks on Methods

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0:00
Hi, my name is Giles Yeo and I'm a geneticist based at the University of Cambridge. This lecture is entitled 'Genomics 101: an Introduction to Sequencing'.
0:11
This is Fred Sanger, who passed away in 2013. He won two Nobel prizes, because one is not enough. In 1958, he won a Nobel Prize in Chemistry for protein sequencing of insulin (that's determining all of the amino acids within the molecule of insulin), but in 1980 he won his second Nobel Prize in Chemistry, which was for nucleic acid sequencing. Winning one Nobel Prize is enough, but winning two Nobel Prizes and having come up with nucleic acid sequencing was a tremendous achievement, so much so that the Sanger Institute down in the south of Cambridge (which played a huge role in the Human Genome Project) is named after Fred Sanger.
0:52
The Sanger sequencing is important to understand, because it forms the basis for how all of the current technologies of sequencing work, even though it's not exactly the Sanger method any more. I thought it would be worthwhile to spend a few minutes explaining how the Sanger sequencing method works. The Sanger sequencing method is a chain termination method, using specific nucleotides called 'dideoxy terminators'. When a dideoxy terminator of A, T, G, or C binds, the DNA strand no longer extends. In other words, when a dideoxy terminator binds you end up with a stop, and that's it, it's stopped. Say you have a fragment of DNA that you want to sequence, and you put in the DNA polymerase and you put in the normal nucleotides, but you titrate in some terminating nucleotides, given Avogadro's number worth of DNA template, suddenly you get a random incorporation of these terminating nucleotides so that you end up with an entire library. Because you start with so many fragments of template DNA, and because there is a random incorporation of the terminating nucleotides, you end up with a library of fragments representing every single nucleotide that is there. The original sequencing technologies used radioactive dideoxy terminators such as A, T, G, or C which were radioactively labelled, so when the terminating nucleotide was incorporated you could see it using X-ray. You had to use four different tubes to distinguish between the radioactivity: an A tube, a T tube, a G tube, a C tube. Once you did the reactions you ran it out on a polyacrylamide gel, which was able to distinguish differing single base-pair differences in length, and you ended up with this ladder. Reading from the bottom up, the sequence would then be reading A-T-G-C-A-T-A-A, and so on. Because you could tell the difference between every single fragment, you were able to read the ladder of the DNA fragments going upwards.