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DNA isolation after digestion with micrococcal nuclease

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DNA isolation after digestion with micrococcal nuclease

Postby havery01 » Wed Dec 01, 2010 12:31 am

You isolate the DNA from an unknown organism and briefly digest it with micrococcal nuclease. You find the following:

a)When the proteins are removed from DNA and electrophoresed you find DNA fragments that band at positions equal to 260 base pairs, 520 base pairs, 780 base pairs etc.

b)The DNA without proteins and completely digested by micrococcal nuclease shows bands equal to mono-nucleotides and dinucleotides.

c)When the proteins without the DNA are electrophoresed there are 2 bands each with a weight of about 26 D.

d)The molar ratio of the two proteins is about 1:1

Explain if these results suggest or do not suggest that this DNA has a nucleosome-like structure. You must include how each piece of information (1-4) helped in your explanation

>What I think: I think yes. It does have a nucleosome-like structure.
a)the length is listed in base pairs, so they are relatively small DNA fragments. The nucleosome core consists of approximately 147 base pairs plus the stretch of linker DNA which varies from 10-80 base pairs. So added together the number is still pretty small, and compared to listed lengths in the question, slightly similar.

b)as far as (b) goes im confused at what kind of bands mono-nucleotides and dinucleotides produce. any kind of lead on how i could find this information out would be wonderful.

c)If the amino acids weight are being measured in daltons i would say that these proteins are small or weigh very little?

d) if the two proteins have a molar ratio of 1:1 then they are very similar? nucleosome structures are alpha helical, and so are the majority of proteins, so does that have anything do do with this question?
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Postby canalon » Wed Dec 01, 2010 1:09 am

a) correct
b) not much bands in most gels used they are very small. Just another way to say that the DNA has been fully degraded back to the nucleotides Probably a few interact in solution to form the dinucleotides.
c) 26 Da seems awfully small for a protein. Even 26 kDa is far from massive (the ladder I use starts at 7kDa, and IIRC a dinucleotide is 11Da[might be wrong, that]) I suspect a typo. But small protein for sure.
d) molar ratio of 1:1 means that for each mole of prot.A you have one mole of prot.B They can be very different, and says nothing about structure.
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Re: DNA isolation after digestion with micrococcal nuclease

Postby havery01 » Wed Dec 01, 2010 1:43 am

I thought it might be a typo too. My professor is famous for those. But it is printed in more places than just that question. maybe i am just not understanding what he means. for example. he says: The amount of charge per unit of mass of DNA is same of all sizes of DNA due to the fact that for each base pair, about 660 D, there are two charged phosphate groups....
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Postby canalon » Wed Dec 01, 2010 5:01 am

First you proved me wrong, I don't know if I should thank you ;) so a pair of nucleotides is 660Da, not 11. So imagine for a protein, 26Da: not realistic.
For your sentence it is perfectly nice and correct: if we assume that every pair is the exact same weight (probably not entirely correct, but reasonable), and that each pair has 2 charges, the amount of charges per unit of weight is constant (1 per 330Da/or 2 per 600Da)
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Postby JackBean » Wed Dec 01, 2010 10:26 am

b) as canalon said, it's just way to say, it's completely degraded, so the lengths shown in a) are due to the proteins, which protect the DNA from degradation ;)

c) I just don't get much, how can you see two bands of the same size? BTW average amino acid weigth is about 130 Da, so 26 Da is like one calcium or two carbons ;)
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Re: DNA isolation after digestion with micrococcal nuclease

Postby havery01 » Thu Dec 02, 2010 4:47 am

The nucleosome is the basic unit of DNA packaging in eukaryotic cells. so would it be safe to say that in part (c) that this information could mean the protein bands weighed so little because the nucleosome structure was such a large part of it?
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Postby JackBean » Mon Nov 28, 2011 4:15 pm

what? That doesn't make sense.
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