You have isolated a novel species of yeast from the dumpster behind Appalachian

Question

You have isolated a novel species of yeast from the dumpster behind Appalachian Mountain Brewery. You would like to sequence the genome of this yeast species for further analysis. Which sequencing method discussed in class would most efficiently provide us with a complete genome sequence? Please justify your answer. Now that we have a complete genome sequence of our yeast species we would like to analyze one of the genes more closely. We find a gene that we predict to encode an alcohol dehydrogenase enzyme - it is roughly 600 bp in size. We name the gene AMB. in order to study the function of the AMB gene, we use PCR to amplify its sequence and then insert it into a plasmid. We want to sequence our now AMB plasmid to make sure that the gene was inserted correctly. Which sequencing method would allow us to most efficiently sequence our plasmid? Please justify your answer.

Explanation / Answer

For complete genome sequencing: A modified form of Sanger's method called Capillary Sequencing Methods used. In capillary sequencing machines, fluoresecnt labelled digested genomic DNA fragments are separated based on size through a long, thin, acrylic-fibre capillary instead of an electrophoresis gel. The various steps are as follows: 1. A sample containing fragments of DNA is injected into the capillary. 2. Electric field is applied, to drive the DNA fragments through the capillary. 3. A fluorescence detecting laser, built into the machine, then shoots through the capillary fibre, causing the coloured tags on the DNA fragments, to fluoresce. Each base terminator is labelled with a different colour: A = Green, C = Blue, G = Yellow and T = Red. 4. The colour of the fluorescent bases is detected by a camera, and recorded by the sequencing machine. 5. The colours of the bases are then displayed on a computer as a graph of different coloured peaks

For sequencing of AMB gene: A modified form of Sanger's method called Pyrosequencing method is used. Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.

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