PCR+enhancer - (Mar/22/2009 )
Is there anyone who can explain me why people use and enhancer solution in a PCR reaction? Wat is the function of that enhancer solution?
-minemin-
minemin on Mar 22 2009, 10:38 AM said:
Is there anyone who can explain me why people use and enhancer solution in a PCR reaction? Wat is the function of that enhancer solution?
you mean additives?
BSA - the plastic surface of the PCR tube can bind to protein such as the DNA polymerase. So the BSA acts to block the plastic surface allowing more DNA polymerase to be active during the reaction
betaine and DMSO - these compounds disrupts the water structure. dsDNA is held together not by hydrogen bonding but by exclusion of the bases due to the formation of the water cage structure. So with a disrupted water structure the melting temperature of the dsDNA is lowered.
Mg 2+ ions - required for activity of DNA polymerase. Also required as dNTP chelate this ion. As concentration of Mg2+ increase, DNA polymerase activity increase, but specificity is reduced.
K+ ion - increase activity of enzyme.
DTT - reducing agent. Helps prevent oxidative damage to DNA polymerase. Gives longer life span to polymerase.
Uracil-DNA Glycosylase - dCTP is suffers thermal deamination producing dUTP. This enzyme removes the dUTP. useful for long range PCR <10kb
-perneseblue-
perneseblue on Mar 22 2009, 12:00 PM said:
minemin on Mar 22 2009, 10:38 AM said:
Is there anyone who can explain me why people use and enhancer solution in a PCR reaction? Wat is the function of that enhancer solution?
you mean additives?
BSA - the plastic surface of the PCR tube can bind to protein such as the DNA polymerase. So the BSA acts to block the plastic surface allowing more DNA polymerase to be active during the reaction
betaine and DMSO - these compounds disrupts the water structure. dsDNA is held together not by hydrogen bonding but by exclusion of the bases due to the formation of the water cage structure. So with a disrupted water structure the melting temperature of the dsDNA is lowered.
Mg 2+ ions - required for activity of DNA polymerase. Also required as dNTP chelate this ion. As concentration of Mg2+ increase, DNA polymerase activity increase, but specificity is reduced.
K+ ion - increase activity of enzyme.
DTT - reducing agent. Helps prevent oxidative damage to DNA polymerase. Gives longer life span to polymerase.
Uracil-DNA Glycosylase - dCTP is suffers thermal deamination producing dUTP. This enzyme removes the dUTP. useful for long range PCR <10kb
No, you can by just an enhancer solution. You do everything like a normal PCR (inclusive with MgSO4) except you put some extra from the enhancer solution.
I'm wondering what the extra function of this solution can be. I think it's for reducting GC rich regions, but I'm not sure. And if it is so, how does it work?
-minemin-
i think this enhancer solution is esentially DMSO.... i always use DMSO for amplification of genomic template DNA very successfull.
-markusda-
markusda on Mar 23 2009, 07:40 AM said:
i think this enhancer solution is esentially DMSO.... i always use DMSO for amplification of genomic template DNA very successfull.
Oh, ok... and do you know how DMSO can enhance your PCR reaction?? What is the reaction mechanism?
-minemin-