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We are characterizing YADH-1 in my biochemistry lab course. Over the course of the first weeks, we harvested E. coli cells, and isolated plasmid DNA via alkaline cell lysis. A previous exam for the course asked the following question concerning plasmid DNA and analysis:

"Is chromosomal DNA more likely to interfere with restriction mapping, with PCR analysis, or equally likely to interfere with both methods?"

Since we're using EcoRI and BamHI, my thought process is that while we can predict the cuts on a circular plasmid, we wouldn't be able to predict all of the cuts on high-molecular weight chromosomal DNA, and the resulting restriction map would be useless. However, PCR can work in a complex mixture of molecules, so even in chromosomal DNA, the primers should be able to find the target sequence. Is the answer therefore that chromosomal DNA is more likely to interfere with restriction mapping?

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Welcome to Biology.SE!

I think you've come to a reasonable conclusion, but not for exactly the right reasons.

The genomes of several strains of Escherichia coli have been sequenced (e.g. https://www.ncbi.nlm.nih.gov/nuccore/U00096) so you could predict the sizes of the fragments you would expect from your digest.

The genome size of E. coli is about 4.6 million base pairs and six-cutters like EcoRI and BamHI cut on average about once every 4096 bp (you should be able to figure out how to come up with that number). From that what would you expect to see if you did a restriction digest on this bacteria's genomic DNA?

Does that help you improve your answer?

(If you have access to the appropriate software, I encourage you to download that sequence and check your conclusions.)

((Also note that the specificity of PCR is dependent on primer selection, so your answer is assuming that the primers were well designed!))

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  • $\begingroup$ Ah, it does. That means I'd get about 1100 different cuts on a map for genomic DNA, and wouldn't be able to conclude if I'd isolated the gene of interest. $\endgroup$ – austintexis Oct 19 '19 at 18:29
  • $\begingroup$ Yes and each of those fragments will be a different size! I found a relevant picture here: semanticscholar.org/paper/… $\endgroup$ – tyersome Oct 19 '19 at 18:38

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