Ligations

[Nashyboyo]

I'm writting my dissertation and i've come accross a mental block and its driving me mad.

 

I did some work in the lab and now i'm writting it up and i've totally forgotten how I come to one conclusion I made. I decided on an appropriate ligation teperature using two simple factors

 

1) the optimum temperature of the ligase was 40 degrees C

2) the laws of thermodynamics

 

I can't remember which law I thought of and its soooooo simple its driving me mad. I'm getting confused over something so trivial can anyone help me out.

 

By decreasing the temperature of a system you lower the random motion of particles which therefore lowers the probability that two reactants come together in the correct orientation for reaction...right ? Therefore increasing the temperature of that system would be favorable for ligation between two DNA molecules right ?

 

I have written down in my notebook "Rondom motion doesn't favour two molecules coming together" WHY DID I WRITE THAT. WAS I RIGHT ?

 

P.S. i'm hungover

[mattbimbo]

hi I.H. post script,

i guess you are thinking about collision theory and reaction specificity. practically i don't know anyone who uses ligases at 40C - the reaction goes too quickly - i prefer overnight in the fridge. i like it slow. i do have a trick which i learnt from a russian 'clone anything' molecular biologist/'catch anything' fisherman and that is to include linear acrylamide in my ligations. can you guess why? i know there are commerical ligase kits which one uses for 5-60mins at room temperature. basically these high efficiency kits just have high amounts of pure enzyme rather than containing an especially efficient enzyme. maybe you are aware that with in vitro reactions with ligase there is considerable inactivation of the ligase?

 

anyway you are sort of RIGHT!

i write sort of - because there is a need to have kinetic energy for the molecules to come together...iigase, ATP, DNA molecules 1 and 2...

and kinetic energy is important for the annealing of the dna molecules...

also for the release of the ligated DNA molecule...

so you are sort of WRONG too!

[Yggdrasil]

Since ligase is a catalyst, the issue here isn't thermodynamics, but kinetics. The rate of the ligation reaction, much like any reaction, is temperature dependent. In general, increasing the temperature of a reaction will increase the rate of the reaction. However, the rate of the ligation reaction is also dependent on the catalyst (the ligase enzyme). Enzymes have a complex three-dimensional structure which is essential to their proper function. (Now here's where the thermodynamics come in) at high temperatures states of high entropy (more disorder) are favored while states of low potential energy are favored at low temperatures. Since a correctly-folded, functional enzyme corresponds to a low potential, low entropy state, it is favored more at lower temperatures. Denatured (i.e. nonfunctional) enzyme corresponds to a high potential, high entropy state, and is favored at high temperature.

 

Therefore, there are two opposing factors at play. The rate of reaction increases with temperature because of the kinetics of chemical reactions. However, the efficiency of the enzyme decreases with temperature, decreasing the rate enhancement by the enzyme. Therefore, the rate of reaction will increase up to a certain temperature when the enzymes start denaturing from the heat, after which the rate of reaction will decrease with temperature. It just so happens that this peak temperature is ~40C for DNA ligase.

[trvlrpsmyrph]

40 degrees centigrade is VERY humid! Try 40 degrees farenheit or lower the centigrade temp to 20.

[mattbimbo]

i don't especially want get into this question again, because it is a bad question, but to write an answer in favour of kinetics and not thermodynamics is potentially misleading.

if something is TEMPERATURE DEPENDENT how can thermodynamics be irrelevant? need i say more?

(but i concur with most of Yggdrasil's reasoning.)

 

and 40C - humid - what nonsense? the enzyme works in solution, ie 100% humidity.

[Bluenoise]

40 degrees centigrade is VERY humid! Try 40 degrees farenheit or lower the centigrade temp to 20.

 

lol

[prion]

Don't know if this helps but the small numbers of ligations I've done have always been in the fridge overnight - I understood that at higher temperatures you were more likely to get unwanted ligation products like plasmids with no insert, multiple inserts, etc. Can't vouch for this though.

 

And protein stability isn't as simple as cold = stable, hot = unstable. They have a temperature of peak stability and it decreases each side of that. Some will cold-denature.

[CharonY]

Actually I think I read somewhere that annealing of the DNA-templates (that is, forming of hydrogen bonds) is a (the?) rate limiting step in this reaction. At high temperatures these bonds are naturally less stable or easier disrupted.

Of course, a host of protocols exist and depending on your templates it might not be essential at all. With sufficient vector and insert e.g. an 1-2 h incubation on the bench usually works. On the other hand I often do o/n incubations in thermos flasks in the fridge for blunt end ligation. Using the thermos flask the temperature decreases more slowly and supposedly increases efficiency...