DNA Extraction

[ender7x77]

OK...I'm a little bit unsure of my anwsers in regards to extracting DNA from peas so I was hoping if anyone could confirm them for me. I looked up a site that had remotely the same procedure if it helps http://learn.genetics.utah.edu/units/activities/extraction/

 

- The meat tenderizer acts as an enzyme such as Bromelain and Papain, which are proteases to cut away proteins (histones) from the DNA allowing for it to unravel. This reactant is important because... (I'm a little stuck on this... I know what histones are, but cannot seem to find anything on why they must be removed in order to unravel the DNA...ideas?)

 

- While the blender separates the pea cells, there still remains the issue of isolating the DNA for extraction, which is achieved by utilizing an emulsifying agent (detergent) to destroy both the cell membrane and the nuclear membrane. Since, an emulsifying agent is amphiphilic (like likes like) and the two membranes consist of lipids and proteins, the detergent will combine with the two in order to destroy the membranes allowing DNA to be extracted.

 

- Since alcohol is less dense than water, it stays afloat on top of water whereas the cell residue settles at the bottom. DNA precipitates when in the presence of alcohol, which means that it is insoluble in alcohol causing for the collection of DNA in the alcohol layer.

 

I know I'm asking for a lot, but even if anyone could just review one of my anwsers it would be greatly appreciated!

[CharonY]

- The meat tenderizer acts as an enzyme such as Bromelain and Papain, which are proteases to cut away proteins (histones) from the DNA allowing for it to unravel. This reactant is important because... (I'm a little stuck on this... I know what histones are, but cannot seem to find anything on why they must be removed in order to unravel the DNA...ideas?)

 

You add proteases to a) clean up your DNA extract from some proteins in general and b) DNA binding proteins in particular. DNA is wound tightly around histones and if this association is not disrupted, you will find protein contamination in your DNA sample.

 

What is the question for the next two parts?

[ecoli]

DNA precipitates when in the presence of alcohol, which means that it is insoluble in alcohol causing for the collection of DNA in the alcohol layer.

I thought the idea behind using alcohol was to precipitate proteins. is that wrong?

[Bluenoise]

I thought the idea behind using alcohol was to precipitate proteins. is that wrong?

 

Some alcohols can percipitate some proteins and all DNA, however in this procedure it is used to percipitate nucleic acids. The proteins wont percipitate because treatment with the protease has already chopped them up into oligiopeptides (which don't percipitate easily), and I doubt an ethanol layer on water can cause significant proteins to percipitate and collect in the interphase.

 

Alternative methods use a buffer that percipitate only proteins (salt, pH various ions etc), which are then removed by centrifugation prior to collection of the DNA. However the method stated in the OP is an at home method so trys to avoid the use of expensive lab equipment.

 

As mentioned above if the histones aren't removed by a method such as protease digestion they will contaminate your DNA. Which is important in downstream applications such as sequencing and in-vitro transription, as they may block the progression of polymerases and other DNA binding enzymes.

(It's best to do a phenol-cholorform extration prior to use to remove everything, however this once again isn't an at home thing.)

[ender7x77]

Thanks a lot everyone! I would have assumed that without removing the histones that the DNA may not have precipitated; also the proteins I would assume to be are those classified as cell residue and would simply go to the bottom because they are more dense than water(?).

 

Sorry, I forgot to list the questions so thanks a lot for anwsering/confirming some of my anwsers. Here are the questions in order of my anwsers:

What is found in the meat tenderizer that is needed for DNA to be extracted?

 

Liquid detergent is an emulsfying agent, why is it needed?

 

Why does the DNA collect in the alcohol layer?

 

Also - The salt added at the beginning (this is not one of my questions) helps in the precipitation of the DNA...? I know it is quite the assumption, but the procedure for DNA extraction seems to be so precise that the addition of salt must be important. Anyways, that was just a thought i was curious about.

 

Thanks again everyone...hope you had a great New Years!

[Bluenoise]

Thanks a lot everyone! I would have assumed that without removing the histones that the DNA may not have precipitated; also the proteins I would assume to be are those classified as cell residue and would simply go to the bottom because they are more dense than water(?).

No they will not percipitate at standard gravity. You need to increase it via centrifugation for this to occur. Brownian motion is stronger than gravity for particles this small.

 

Also - The salt added at the beginning (this is not one of my questions) helps in the precipitation of the DNA...? I know it is quite the assumption, but the procedure for DNA extraction seems to be so precise that the addition of salt must be important. Anyways, that was just a thought i was curious about.

 

Thanks again everyone...hope you had a great New Years!

 

Yes salt is necessary to percipiate DNA since a positive counter ion is needed.

However in most cases this much salt is already present and the addition of more is not necessary.

[ender7x77]

I thought you said that centrifugation was in the method whee the proteins precipitated...perhaps I misinterpreted. In my experiment the DNA precipitated. Anyways, thanks though I'm not making the connection with your last post.. the positive counter ion is above my knowledge.

[Bluenoise]

Righty-O!

You seem to have a good grasp of the procedure.

[ender7x77]

thanks. Was my assumption of the emulsfying agents effect on the pea cell correct? Out of all the anwsers I've been most dubious about that anwser because I've never been comfortable with what goes on with amphiphilies. I know they have a tail and a head end, which one represents the hyrophobic side and the other the hydrophilic side, but my knowledge doesn't really extend much further than that. If you could explain or confirm that would be great especially since exams are closing in.

[Bluenoise]

Yes you're correct. But I don't like the use of the word destroy. The detergent (emulsfying agent) disolves the membrane. Since the DNA is encapsulated both by the cellular membrance and nucleaus you need to break appart the membranes into small bits so it can get out.

 

 

Oh go here this will explain it. Very nice page.

 

http://learn.genetics.utah.edu/units/activities/extraction/detergent.cfm

[ender7x77]

Thanks again... I guess I know more than I think I know. I guess the last thing I'm uncertain about is what attaches to the tail end and what attaches to the head end. I read that page although it strengthened what I wrote and what you confirmed, it doesn't necessarily say where the protein and lipids attach to in order to dissolve the membranes. Thanks for continuing in helping me.

[Bluenoise]

attaches?

Well the detergent tails "attach" to the proteins or tails of the cell membrane lipids.

 

While the heads point out into the water as they're hydrophilic.

[ender7x77]

Ok..sorry wrong choice of a word. I'm just really unsure of what happens to both of the ends in order for them to dissolve the membranes. I apologize for all the questions.