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For a large genome (say millions of base pairs) -- we split the genome into fragments < 1000bp that are sequenced and can then be put back together computationally.

My understanding is you use some sort of shearing technique to get the DNA into fragments as in: https://en.wikipedia.org/wiki/DNA_fragmentation

Now you have a vat full of random fragments -- how do you sanger sequence these?

From what I can tell if you took the solution with the sheared DNA and ran sanger sequencing you'd get a bunch mixed of fragments mixed together resulting in a lot of noise ... you'd basically get all the runs averaged together. Don't you need to isolate each fragment before running Sanger sequencing?

How is this done?

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In the original method, one would clone all of the resulting fragments into a suitable vector, and transform the collection into a bacterial host, to create a so-called library. By plating the library clones under appropriate conditions you would have a collection of so-called clones, and each clone would be randomly selected and sequenced using the Sanger technique.

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  • $\begingroup$ So if I understand correctly: Step 1: inject the fragmented plasmids into vectors. Step 2: Pick out an individual vector (e.g a single e-coli) and put it on a petri dish (this represents a single fragment) Step 3: Allow the individual vector to replicate (and thus replicate your fragment) Step 4: Take the culture of vectors, lyse the cells and extract the DNA. Step 5: Run Sanger sequencing $\endgroup$ – user491880 Aug 4 '18 at 19:14
  • $\begingroup$ Step 0: Prepare double-stranded DNA cloning vector (usually a circular DNA Plasmid) by digesting, or cutting, it with a site-specific type II restriction enzyme, or endonuclease, and then possibly purify this uniform DNA fragment. Step 0.5: Mix the purified, linearized, cloning vector fragment with the suitably prepared sheared DNA fragments (that you want to sequence), and add ATP, buffer, and an enzyme named DNA ligase. Specifically, DNA ligase encoded by the genome of bacteriophage T4. Step 0.9: Treat log phase E. coli cells with divalent cations, on ice (making them competent). $\endgroup$ – mdperry Aug 5 '18 at 16:13
  • $\begingroup$ Step 1: Mix a small amount of the ligated DNA with the competent cells, give them a heat pulse, add some broth and grow them at 37 briefly, and plate aliquots on LB Plates containing a drug that will select for the rare bacterial cells that have taken up one of the foreign recombinant plasmids (from the DNA ligase reaction). 99.99 % of the bacterial cells will be killed by the drug in the plates, but cells that have taken up a viable copy of the cloning vector will express its selectable marker (a drug resistance gene) and be able to grow, yielding a bacterial colony on the plate). $\endgroup$ – mdperry Aug 5 '18 at 16:19
  • $\begingroup$ Step 2: Pick individual colonies, grow them in broth. Now continue with your Step 3 and Step 4 $\endgroup$ – mdperry Aug 5 '18 at 16:20

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