If you attempted to knock out a yeast gene that is 3.0 kb in length by construct

Question

If you attempted to knock out a yeast gene that is 3.0 kb in length by constructing a linear DNA fragment that replaces the middle 2.5 kb of the coding sequence of the gene with a selectable marker that is 1.5 kb in length and transforming this fragment into yeast( assume you get transformants that survive the selection, which of the following techniques could you use to determine whether the fragment had intergraded into the chromosome in a manner that knocked out your gene?Microarray, PCR, DNA sequencing, Northern blot, and Southern blot Briefly describe all that could be used.

Explanation / Answer

To confirm whether a given gene is knocked-out in yeast

1. A microarray is not suitable as it would generate genome-wide transcription data

2. PCR with primers specific to the selectable marker would discriminate between WT and the mutant.

3. DNA sequencing using flanking primers also will give information about the entire region whether the knock-out strategy worked or not.

4. Northern blot specific to WT gene will show whether the given gene expression is reduced/diminished or not (indirect confirmation method).

5. Southern blot with gene-specific or insert specific probe will give good information about the WT/Knock out status.

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