You wish to perform whole-genome shotgun sequencing of a new species of yeast t

Question

You wish to perform whole-genome shotgun sequencing of a new species of yeast t you have identified growing in the spine of your Genetics textbook. Consider the follwoing steps that you would perform. Mix genomic DNA fragments from the yeast with a plasmid vector digested with BamHI and add DNA ligase. Isolate DNA from bacterial clolonies and carry out dideoxy sequencing on each them. Use a computer to 'assemble' the DNA sequences into the order they were in the intact genome. Isolate genomic DNA from the organism and digest it with a restriction enzyme sl as BamHI. Transform the DNA mixture into bacteria and plate on plates containing ampicillin (assume the vector carries ampicillin resistance gene). Which is the correct order for you to perform these steps in order to sequence the r species' genome? a) 2 - 4 - 1 - 5 - 3 b) 1 - 4 - 5 - 2 - 3 c) 3 - 2 - 4 - 1 - 5 d) 2 - 1 - 4 - 5 - 3 e) 4 - 1 - 5 - 2 - 3

Explanation / Answer

Answer is (e) 4-1-5-2-3

4-First isolate the gDNA from the yeast organism and digest it with BamHI (you can use any enzyme of your choice). A six cutter would be preferable so that larger fragments will be obtained (as there is 1 in 46 chances of this site being in the genome as opposed to a 4 cutter which would be found once in 44 times.

1-Mix the digested gDNA fragments with a vector that has been cut with the same enzyme. This is to allow the ends to have compatible ends with the vector. Add DNA ligase to join the DNA fragments to the vector.

5- Transform the ligated DNA into competent bacteria and plate these cells on media containing ampicillin (assume the vector carries the ampicillin resistance gene).

2-Isolate DNA from each of the bacterial colonies that come up on the plates and carry out dideoxy sequencing. That is the fragments that have been cloned into the vectors are sequenced.

3- Use a computer to assemble the DNA sequences in the order they were in the intact yeast genome.

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