Standarization of HRM for methylation - (Sep/04/2010 )
i am trying to standardize HRM ( high resolution melting ) for methylation studies but are i am facing following problems;
i am getting clear specific bands in conventional PCR but there is no amplification in real time pcr , i have even tried at different temperatures , at some temperatures there is amplification and melting peaks but only for primer dimmers and nonspecific band and very little amplification for specific band.
anyone having knowledge about HRM , kindly help.
What are your HRM/PCR conditions? (primer concentration, MgCl2 concentration, template concentration, anealing temperature and time)
HRM is more inhibiting than normal PCR (even more than SYBR). If you have some SYBR mix, try it, if it amplifies something and try to optimise temperature on your real-time machine. If doesn't, play with the concentration of the primers. When you got the right temperature, switch to HRM mix and optimise MgCl2 concentration (the more you add, the less stringent the reaction will be).
Thanks for the suggestions.
the pcr conditions in for conventional pcr are:
template DNA = 80ng
primer conc = 0.5 Ám
MgCl2 conc. = 3mM
Anneling Temperature = 58c
the pcr conditions in for Real Time pcr are:
template DNA = 80ng-10ng ( i have tried serial dilution of template DNA , amplification is seen little bit with increased dilution but Cp value is above 30)
primer conc = 0.125 Ám
MgCl2 conc. = 3mM
Anneling Temperature = 58C and 56C both ( with better but still very low amplification at 56C along with primer dimmers and nonspecific peaks.
please help me to sort out this problem , what further changes should be done to standardize HRM , how to identify specific peak among multiple peaks .
I've got only one successful optimization behind me and now I'm struggling with a second one.
Manual for my HRM mix (Roche) recomends:
template 5-30ng per 20ul reaction (I used 12ng for 10ul)
Cp < 30
primer concentration 0.1 - 0.3 uM (starting with 0.2, worked first time)
short amplicons (100-250 bp)
MgCl2 1 - 3.5 mM
So, if you have low amplification, I would use 30ng and 0.3 uM primers first at the 58 deg, to see I you can get under 30. If not, then probably try higher concentration of primers or template, but that's outside the recomended limits, so it's hard to say what it will do. Use some template you have in excess for this, like WT or something.
If you get fine amplification, then check for the specifity, dimers, multiple peaks, first I would try dilution series of MgCl2 with a step of 0.5 mM per reaction in the recomended range, 3mM is rather high, so maybe decreasing it would free you of the multiple peaks. Annealing of the primers is Mg and temperature dependent, so you may optimize for temperature or MgCl2 concentration (but you can do MgCl2 range in one reaction). You may also decrease the annealing time.
What dye are you using? When working with SYTO9 I usually have to lower the annealing temperature from 1-2 degrees compared to conventional PCR. If your realtime PCR doesn`t work you can also amplify your template by conventional PCR, add the dye afterwards to your pcr product and do an additional denaturation-renaturation step prior to melting.