And also for RT-LAMP, the temperature is between 60 and 65 C degree. Why so high temperature can be used to reverse transcription? Normally, RT is done at 37C.
Edited by xxcici, 08 March 2013 - 06:17 AM.
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How double stranded DNA becomes single stranded in LAMP assay?
Started by xxcici, Mar 08 2013 05:39 AM
3 replies to this topic
#1
xxcici
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Posted 08 March 2013 - 05:39 AM
LAMP reaction is performed at constant temperature without heat denaturing of double stranded DNA. So, how the double stranded DNA can become the single stranded for primer to anneal?
And also for RT-LAMP, the temperature is between 60 and 65 C degree. Why so high temperature can be used to reverse transcription? Normally, RT is done at 37C.
And also for RT-LAMP, the temperature is between 60 and 65 C degree. Why so high temperature can be used to reverse transcription? Normally, RT is done at 37C.
#2
bob1
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Posted 09 March 2013 - 12:23 AM
I don't know the specifics of this assay, but you can alter the denaturation temperature and annealing temperature by different concentrations of salts.
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Posted 09 March 2013 - 08:33 AM
It's even on Wikipedia, the polymerase has a strand displacement activity.
The whole reaction is bit more complicated though as I understand from the original paper.
The whole reaction is bit more complicated though as I understand from the original paper.
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Posted 09 March 2013 - 09:09 AM
Thanks. I searched that it is because DNA stays at a thermodynamic equilibrium state at 65 C. So, double stranded DNA and single stranded DNA stay in an equilibrium situation a+b=ab. In this situation, the primer can anneal with the double stranded DNA causing the the other strand of the template to break.
AMV reverse transcriptase can be used up to 70C.
AMV reverse transcriptase can be used up to 70C.
Edited by xxcici, 09 March 2013 - 09:24 AM.
