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Identify the nucleic acid!

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Identify the nucleic acid!

Postby vaibhavsmarty » Sat Feb 16, 2008 5:15 am

A nucleic acid sample is resistant to digestion with lambda exonuclease.When heated it does not show typical melting curve of a linear double stranded DNA.On CsCl-ethidium bromide equilibrium density centrifugation it settles at the bottom of the centrifuge tube.The nucleic acid is:
a/ccc pBR322
b/Bacteriophage P22 DNA
c/rRNA
d/RF II M13 DNA
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Re: Identify the nucleic acid!

Postby Cat » Sun Feb 17, 2008 12:20 am

I would say it's Bacteriophage P22, but not 100% sure.
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Re: Identify the nucleic acid!

Postby blcr11 » Sun Feb 17, 2008 12:16 pm

My bet would be on rRNA. The exonuclease in question acts exclusively on ds DNA. The insensitivity to it almost automatically rules out all the other choices because they are all ds DNAs: pBR322 is a standard plasmid; Phage P22 DNA is ds; M13 RF DNA (RF stands for “replicative” form, though I’m not quite sure what the II is for) is a ds circle, although the infective DNA is ss and would be resistant to lambda exonuclease. rRNA should be resistant to the exonuclease, does not have a typical melting curve, and is very dense compared to typical duplex DNA. If you’ve never run a CsCl gradient before, consider yourself lucky. The intense staining at the bottom of the tube is due to all the rRNA that pellets there.
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Re: Identify the nucleic acid!

Postby blcr11 » Sun Feb 17, 2008 1:23 pm

Concerning CsCl gradients, I should also point out that you're supposed to treat your sample with RNAse first to avoid all that RNA contamination--but if you happen to forget, or underdo it, then you see a lot of staining at the bottom. You will still see some staining due to RNA even if you've done the RNAse treatment, but not nearly so much.
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Postby Cat » Wed Feb 20, 2008 6:40 pm

This question is unfair. 1) resistant to digestion with lambda exonuclease: lambda exonuclease has 5' to 3' activity on double stranded DNA so all choices given would be resistant since 3 of them are circular DNA and 1 is RNA; 2) it does not show typical melting curve of a linear double stranded DNA: again non of the choices are linear double stranded DNA; 3) On CsCl-ethidium bromide equilibrium density centrifugation it settles at the bottom of the centrifuge tube: only two choices can be ruled out - a/ccc pBR322 and d/RF II M13 DNA - because they are relatively small (4361 bp and 6407 bp) while Bacteriophage P22 DNA is 41661 bp and likely to precipitate. rRNA will precipitate as well (if untreated with RNase) so it could be the answer, but since CsCl-ethidium bromide equilibrium density centrifugation method is designed to separate DNA, I am inclined to go with the most likely DNA choice.
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Re: Identify the nucleic acid!

Postby blcr11 » Wed Feb 20, 2008 9:11 pm

I don’t disagree with you that much, Cat. I’d still bet on rRNA, though on further reflection, the question may not be answerable with the information given. I see that lambda exonuclease cannot initiate digestion from a nick or a gap (although it does have very weak activity against ssDNA or so says the descriptive blurb in the NEB catalog), so if it cannot work on circles at all, then you are right about it not being a fair question.

I think you can get reasonable melting profiles out of circular DNA. I don’t know how different they look compared to authentic linear DNA. And it’s been awhile since I’ve thought about phage DNA of any kind, so I’m not sure if the packaged form of P22 DNA is linear or circular. I know it is duplex. If it is linear, it should be susceptible to lambda exonuclease. Certainly once inside the cell it becomes a circle fairly soon, if it wasn’t circular to begin with. Once replication begins, the replicated DNA is at least transiently linear, but in what form it gets packaged? dunno. But if circles are completely resistant to lambda exonuclease, then two of the three forms of pBR322 should also be resistant, which complicates any interpretation again.

With respect to the CsCl gradients, it isn’t that larger DNAs or RNAs “precipitate” to the bottom of the tube. RNAs are fairly dense. They are perfectly intact in CsCl, but are just more dense than the densest part of the gradient, so they go all the way through it. You can band lambda phage and P22 DNA on a CsCl gradient.You can even band E. coli chromosomal DNA on CsCl and the E. coli chromosome has over 6 million bases, so size per se isn’t a problem. None of the DNAs listed should pellet to the bottom of a typical CsCl gradient, while that is where I expect to find RNAs.

Assuming that circles are susceptible (enough) to lambda exonuclease, then I still go with rRNA. But if the enzyme has an absolute requirement for linear DNA, then you can’t answer the question with the information given.
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