What Are the Benefits of Using PCR Additives in DNA Amplification?

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Discussion Overview

The discussion focuses on the various PCR (polymerase chain reaction) additives and their potential benefits in DNA amplification. Participants explore the types of additives, their roles, and the conditions under which they may be beneficial, particularly in relation to specific DNA sequences and structures.

Discussion Character

  • Exploratory
  • Technical explanation
  • Debate/contested

Main Points Raised

  • Some participants inquire about the specific types of PCR additives and their mechanisms for improving the reaction, mentioning examples like DMSO, Tween, Formamide, Glycerol, and (NH4)2SO4.
  • One participant expresses surprise at the existence of PCR additives, noting that their PCR reactions have worked fine without them, but questions the necessity of certain additives used in their own experiments.
  • There is a suggestion that Glycerol may act as an oil to prevent evaporation during the PCR cycles.
  • Another participant asserts that PCR does not always work without additives, citing issues with primer stability and secondary structures in DNA as reasons for their use.
  • Some participants mention that additives like glycerol, DMSO, and betaine can be particularly effective when dealing with high GC content in DNA, and that MgSO4 can replace MgCl2 under these conditions to help destabilize secondary structures.
  • DMSO and formamide are identified as polar solvents that may assist in solvation of denatured DNA, while the roles of Tween and spermidine are questioned.
  • Ammonium sulfate is speculated to function through a salting-in effect.

Areas of Agreement / Disagreement

Participants express a mix of curiosity and skepticism regarding the necessity and effectiveness of PCR additives. There is no consensus on their roles or the conditions under which they are beneficial, indicating multiple competing views remain.

Contextual Notes

Participants mention specific conditions such as high GC content and the stability of primers, but do not resolve the implications of these factors on the effectiveness of PCR additives.

Monique
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OK, let's get some molecular biology in here. I would really like to know exactly how many PCR additives there are and why they would improve the reaction.

I am talking about reagents such as DMSO, Tween, Formamide, Glycerol, spermazin?, (NH4)2SO4 etc.


for those who don't know: PCR (polymerase chain reaction) is a technique that linearily or exponentially amplifies DNA, such as specific genes or 'just' the whole genome.
 
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Wow!

There are PCR additives? Why? Seems to work fine without them doesn't it?

I did PCR today, and we used the usuall stuff (all of the nucleotides, Forawrd primer, reverse primer, Taq DNA polymerase..)

Oh, there they are...
we used Tris HCL pH 8.8, MgCl2 and... here it is : (NH4)2SO4

I ahve no idea what those three additives do. Obviously, MgCl2 is a salt, it probably does some salt binding thing with the DNA...but to what effect I don't know.

Thanks for posting this, I will ahve to ask why they are in there now.
 
Would the Glycerol count as an oil? I know we added oil to stop the water from evaporating as it is heated through the cycles.
 
Originally posted by Another God
Wow!

There are PCR additives? Why? Seems to work fine without them doesn't it?

There sure are And no, PCRs don't always work without them.. I have run into this problem the hard way. It is true that the standars PCR consists of the ingredients you describe.. but sometimes the primers are unstable on the sequence or the sequence is rich in secondary structures, that is when the additives come in.

Oh! I have to attend an seminar.. be back later.
 
Additives such as glycerol, DMSO, betaine are very effective (you need luck as well), when your DNA has a high GC region. MgSO4 can be replacing MgCl2 when your GC porcentage is high due to it helps to destabilize secondary structures in DNA.
There should be more additives...let me think
 
DMSO and formamide (N,N'-dimethylformamide, right?) are polar solvents, they'd help solvate denatured DNA. So would glycerol. Tween and spermazine? Never heard of them. Is the last a polyamine like spermidine? With a hydrazine linkage? Ammonium sulfate I imagine would just be a salting in efffect.
 

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