Anealing long oligos in complex libraries

[Bluenoise]

Let say you fragment genomic DNA into around 500bp fragments. Now lets say you denature these fragments by heat or whatever. How would you go about ensuring that as many fragments as possible anneal to their complementary strand. Now obviously as you'll likely have 1000s of times genomic coverage each fragment will have 1000s available that it can consider complementary but it will have about 10 000 - 100 000 times that are not considered complementary.

 

Conventional wisdom suggest that the best solution is to just mix them together in a buffer of moderate salt concentration heat a beaker to around 95C drop the tube containing the oligos in and let it slowly cool to at least 65C.

 

However is there a better way? I reallised that annealing oligos is a pretty simple thing. However, usually one considers small oligos and rarely more than 2-4 species. Finding the optimum match for each becomes more tricky when you have long ones and a 100 000 different species.

Maybe the best thing to do it just greatly increase the time it take the temperature to drop?

[foodchain]

Let say you fragment genomic DNA into around 500bp fragments. Now lets say you denature these fragments by heat or whatever. How would you go about ensuring that as many fragments as possible anneal to their complementary strand. Now obviously as you'll likely have 1000s of times genomic coverage each fragment will have 1000s available that it can consider complementary but it will have about 10 000 - 100 000 times that are not considered complementary.

 

Conventional wisdom suggest that the best solution is to just mix them together in a buffer of moderate salt concentration heat a beaker to around 95C drop the tube containing the oligos in and let it slowly cool to at least 65C.

 

However is there a better way? I reallised that annealing oligos is a pretty simple thing. However, usually one considers small oligos and rarely more than 2-4 species. Finding the optimum match for each becomes more tricky when you have long ones and a 100 000 different species.

Maybe the best thing to do it just greatly increase the time it take the temperature to drop?

 

From what I know, I don’t know really. I don’t know how playing with the temp will work unless you plan to take advantage of that, whatever that may be, as it occurs or something. From how you worded it to my understanding of it, basically it sounds like you want to dissolve the human genome and then build it back up exactly in some fashion, I don’t know how you would do that really, I think a great majority of it would already have to be known then you would have to have some rather precision technology or what not to take advantage of such.

[foodchain]

Thinking about the topic I simply don’t understand why biologists or scientists simply don’t attempt to find a way to use DNA polymerase for such. Really I guess is what I am getting at is already existing biological mechanisms exist that probably could get people everything they want to know, but I don’t know if anyone’s actually worked at "farming" such enzymes or found a way to put them to work or use the process in action in a cell to obtain a result, such as a single strand of DNA or what not. I mean I know PCR exists, but I don’t really consider that the same.

[Paralith]

Conventional wisdom suggest that the best solution is to just mix them together in a buffer of moderate salt concentration heat a beaker to around 95C drop the tube containing the oligos in and let it slowly cool to at least 65C.

 

However is there a better way? I reallised that annealing oligos is a pretty simple thing. However, usually one considers small oligos and rarely more than 2-4 species. Finding the optimum match for each becomes more tricky when you have long ones and a 100 000 different species.

Maybe the best thing to do it just greatly increase the time it take the temperature to drop?

 

I'm not an expert on the subject, but that sounds like a good idea to me. Touch down PCR uses a similar technique where you start the annealing temperature very high and slowly work it down, to make as much of the primer bind as specifically as possible to the template DNA. I really don't know how else you could increase the specificity, other than trying to avoid mixing up that many different sequences in the first place =p

 

Thinking about the topic I simply don’t understand why biologists or scientists simply don’t attempt to find a way to use DNA polymerase for such. Really I guess is what I am getting at is already existing biological mechanisms exist that probably could get people everything they want to know' date=' but I don’t know if anyone’s actually worked at "farming" such enzymes or found a way to put them to work or use the process in action in a cell to obtain a result, such as a single strand of DNA or what not. I mean I know PCR exists, but I don’t really consider that the same.[/quote']

 

DNA polymerase is not responsible for binding together the two DNA strands. DNA polymerase only adds on nucleotides to a single DNA strand. Largely by virtue of proximity, the newly formed complementary DNA strand can easily hybridize to the template. But if you're talking about a whole bunch of different single DNA strands all mixed up together, and trying to get the right complementary strands to find each other - well, that's not really a situation you have naturally, and I don't think there are any enzymes that can really aid that process.

[Bluenoise]

Touchdown PCR is very simular to what I'm getting at except unfortunatley it still deals with rather short sequences. I think I'll just have to see how it works out before coming to any conclusions. I figure letting the temp drop from 95-65 in 20-30 mins should be adequate. I hope these elevated temperatures for long periods dont degrade my DNA too badly.

Hmmm anyone know the temperature that DNA starts to anneal at? I know 92-95 is a good bet for denaturing it all but there is likely a gap below that where it doesn't start to anneal.

 

Actually now that I think of it the best approach maybe to use chemical conditions. I can denature my DNA in 0.25 N NaOH and then slowly renature it with the addition of drop wise 3M NaOAc to a 1/10 final concentration. Though the problem here is that I don't exactley know how quickly it anneals under such conditions.