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

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SUMMARY

The discussion focuses on the benefits of using PCR additives in DNA amplification, specifically mentioning reagents such as DMSO, Tween, Formamide, Glycerol, and (NH4)2SO4. These additives enhance PCR reactions by stabilizing primers, solubilizing denatured DNA, and addressing issues related to high GC content in sequences. The conversation highlights that while standard PCR can function without these additives, they are crucial for improving reaction efficiency and overcoming challenges posed by secondary structures in DNA.

PREREQUISITES
  • Understanding of PCR (Polymerase Chain Reaction) fundamentals
  • Familiarity with common PCR reagents such as Taq DNA polymerase and primers
  • Knowledge of DNA structure and secondary structures
  • Basic molecular biology concepts related to DNA amplification
NEXT STEPS
  • Research the role of DMSO in PCR reactions
  • Explore the effects of glycerol on DNA stability during amplification
  • Investigate the use of MgSO4 as a substitute for MgCl2 in high GC content sequences
  • Learn about the mechanisms of PCR additives like Tween and Formamide
USEFUL FOR

Molecular biologists, genetic researchers, and laboratory technicians involved in DNA amplification and optimization of PCR protocols.

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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