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Nested PCR


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2 replies to this topic

#1 RKA

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Posted 20 January 2010 - 04:27 AM

how this works for methylation analysis ? how the 2 pairs of primers can be made for a region of interest ? please help me ...

#2 Spliffeh

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Posted 03 February 2010 - 07:32 AM

Hy, I am a bit in same situation as you.

I haven't been able to design primers yet. But the following might be good lecture to start with.

Methods Mol Biol. 2009;507:305-23.
Methylation-specific PCR.
Licchesi JD, Herman JG.
Cancer Biology Program, Sidney Kimmel Comprehensive Cancer Center Johns Hopkins, Baltimore, MD, USA.


Good luck:)

#3 pcrman

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Posted 17 February 2010 - 09:37 PM

Nested PCR allows you to obtain enough product for analysis (gel visualization, sequencing, cloning), or to save your precious modified DNA for more PCRs. For BSP, you can use the same pair to run two rounds of PCR to obtain enough product for sequencing. For MSP, you should have three pairs of primers:

First PCR using Universial primers - only amplify modified DNA

2nd PCR using M primers to amplify methylation DNA, U primers to amplify unmethylated DNA.




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