Student query: gene sequencing

James has asked: in gene sequencing, since the dd_PP will have made a cut every base, in order for us to read the sequence, how is it possible for us to read it using electrophoresis when the distance between these bases will be around 0.01 of a nm?

I had to check this one with a colleague, as I’m certainly no expert in this particular field! He reminded me that in fact there’s no cutting involved in sequencing. Instead, the different-sized fragments of DNA necessary for sequencing are generated by incomplete synthesis, using a template DNA strand and dideoxy terminator nucleotides. While the dideoxynucleotides can be added to a growing DNA strand, their chemical structure means that no further nucleotides can be added after them. This means that synthesis of the DNA strand stops at that point. So the end-product of the process will be a large number of DNA strands, differing in length by a single nucleotide.

The electrophoresis necessary to separate these different-length strands, to allow the base sequence to be read, is carried out on acrylamide capillaries (rather than the gels you are probably familiar with), which are sensitive enough to resolve single nucleotides. There’s a nice graphic of the process here, along with a much more extensive explanation (and, at the end, some thought-provoking questions about the ethics of sequencing genomes).

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