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Name: Ashley
Status: Student
Age: 18
Location: N/A
Country: N/A
Date: 2001


Question:
When conducting an experiment where you extract plasmid DNA from a cell and insert it into an ampicillian resistant cell, and if we kept the experiment to extract the plasmid the same what would happen is RNA was the transforming principle and not DNA? would you get the same result?



Replies:
Dear Ashley,

The answer to your question depends upon the purity of your extracted plasmid DNA. If you're certain that there is little or no RNA contamination of your plasmid DNA extract, then you would not expect to obtain any ampicillin-resistant transformants if RNA were actually the transforming principle. This level of purity would probably be most easily attained by RNase digestion of the plasmid DNA prior to transformation to degrade residual RNA.

If your plasmid extract contains a mixture of DNA & RNA, you wouldn't be able to determine which one is actually the transforming principle. But if you were to obtain ampicillin-resistant transformants from a DNA/RNA mixture that had been digested with DNase to degrade the DNA, it would be a good indication that the RNA was actually the transforming principle. Bear in mind that these experiments alone would not be definitive, but could be used to design follow-up studies to determine whether DNA or RNA was actually the transforming principle.

Jeff Buzby, Ph.D.


Dear Ashley,

Your question is not very clear. If your plasmid contains an ampicillan resistance gene, you can not select for transformants when you transform it into cells already resistant. Is that your question? The protocol for extracting plasmid DNA is not the same as that for extracting RNA. You can not transform bacteria with RNA (as far as I know), even if you were able to get the RNA in the cell, it could not replicate (unless it is an RNA virus) nor could it recombine into the DNA of the cell.

If this does not answer your question, please rephrase.

Trudy Wassenaar


Let us back up a little. In the first experiment, you are putting DNA that contains at least two important genes into ampicillan-SENSITIVE cells. The first gene (_ori_) is the origin of replication, that allows the plasmid to replicate in the cells, and the second is the ampicillan-resistance gene so that the transformed cells can be selected for on media containing ampicillan.

So, if you attempted to transform with RNA, you would run into (at least) one major problem: How to have the RNA get changed back into DNA so it could replicate and be present in all the succeeding generations. After all, even if you could get RNA with the above two genes inserted into a cell, that is only ONE cell, not the millions you want to grow up.

All that being said, I think it is a moot point; I know of no successful attempts to transform using RNA. While some types of cells contain enzymes that can convert RNA to DNA, bacteria don't appear to possess these. Hope this helps.

Paul Mahoney, Ph.D.



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