Hello, I am trying to introduce a method using microsatellite analysis to detect uneven X chromosome inactivation. I am using four pairs of primers for four different genes. I do standart single-plex PCR for all pairs before fragment analysis. Unfortunately two of the fragments repeatedly show split peaks and one has several sttuter peaks. One hovewer seems to be fine.
I have tried a couple different polymerases (Takara HS DNA with and without GC enhancer, JumpStart Taq ReadyMix-Sigma Aldrich, HotStar Taq Ready Mix-Qiagen) - all with the same result. Altering melting temperature, elongation time and temperature and number of cycles produces the same result. Other fragmentation analyses using the same protocol work fine so it should not be a problem with the instrument.
Is there anything I can try short of ordering yet another polymerase?
I do not think that the polymerase makes much difference ( except those that add an A to the amplimer) and that most of the structural amplifications that you show are caused by the enzyme slippage in the amplified sequence. I might make an exception for the split peak at 374.98 which could just be over amplification and the signal is too strong and the spectrophotometer is out of its range and clipping the signal to make it fit the electropherogram scale.
Possibly running fewer cycles might minimise the problem but I do not think that redesigning primers or changing polymerase will make any difference so if you really cannot work with structured amplifications then one way could be to measure different loci
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