principle of error-prone PCR???

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feixiaorou
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principle of error-prone PCR???

Post by feixiaorou » Tue Sep 30, 2014 1:23 am

Hello guys, recently I was told by a classmate that Mn2+ ions is the key of error-prone PCR. I checked on the internet and I got nothing. I always thought the low-fidelity PCR enzyme is the reason leading to the error matches of basicpair.
I am now confused, which one is correct? If I am wrong, please be kind explain the principle of how Mn2+ working. Attaching the links of involved websites would be so helpful.

thank you so much~~~~~~~~~~~~~~~~~~
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JackBean
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Post by JackBean » Tue Sep 30, 2014 8:19 am

I've never heard about Mn in PCR (which doesn't mean it's not used) and to my knowledge they always provide magnesium, but there are proteins which work better with one or another (e.g. pyrophosphatase is specific for pyrophosphate with one metal while with the other it can cleave also other substrates as ATP etc.), the reason probably being the size of the metal ions and other properties.
http://www.biolib.cz/en/main/

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feixiaorou
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Post by feixiaorou » Wed Oct 01, 2014 3:51 am

Thanks so much for your answer. But how exactly the size of Mn ions affect the Taq enzyme? Did they changes the fourth-level structure of the Taq enzyme, such as combine with the regulatory subunit to control the activity, or something else. I mean, it is obviously not the charges, Mn2+ ions carry two positive as well as magesium; plus, Mn2+ ions have exactly the same function with Mg2+, to active enzymes. So I am thinking that Mg2+ & Mn2+ combined to maximum the Taq enzyme activities, leave it no time to proofread.
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Cat
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Post by Cat » Sun Oct 12, 2014 7:39 pm

I don't know about the effect on the enzyme. However, Mn stabilizes double stranded DNA and from practical PCR standpoint - excessive magnesium allows for annealing and elongation of mismatched primers.

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Re: principle of error-prone PCR???

Post by feixiaorou » Mon Oct 13, 2014 3:05 am

@cat
thanks, learned~~~
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