MSP Primer design - (Feb/17/2015 )
Hi all!
I am trying to design MSP primers for human BCL11A gene based on the DNA sequences from the CpG Islands - 115 & 106 prior the promoter region and 105 towards the end of the gene.
I have attached the Excel file with all the primer sequences for these regions. The graphic representation from the result aligned the primer with input sequences and I double checked the parameters of the primers. However, I couldn't see any bands after running the PCR. The PCR conditions that I used are:
Mixture: 1microl primer, 10microl master mix, 7microl deionised water and 2microl genomic DNA
PCR:
5 mins at 95oC
30sec at 95oC
30sec at 55oC (chosen based on the primer pair with the lowest average Tm)
30sec at 68oC
(Repeat step 2-4 for 40 cycles)
Another question I have is when you are preparing for PCR, do you mix both unmethylated and methylated primer pairs in the same sample mixture but separately?
I am clueless why the PCR's not working. Please help!!!!!!! And thanks a bunch!
The primer pairs are run separately. Are you able to amplify the unconverted DNA with your methylated primers? You need to get that working before even thinking about working with converted DNA. The volumes you give in your PCR setup tell little without knowing the concentrations. What master mix are you using? I'd recommend a Taq based one rather than an expensive high fidelity one. High fidelity ones won't amplify uracil containing templates (which your converted DNA will contain), and Taq is more forgiving in many ways. Have you considered nested or semi-nested PCR?
I set up the PCR using Thermo Extensor Long PCR Reddy Master Mix and with varying concentration of genomic DNA, ranging from 0.6picog to 60picog. However, I was unable to amplify the unconverted DNA with methylated nor unmethylated primers. Another confusion of mine is when I tried to blast the unmethylated primers, I got results from other chromosomes other than my target DNA. I thought the unmethylated primers was designed based on the original DNA and methylated primers on bisulfide converted DNA? Or are both primer pairs designed based on the differences in cytosine/uracil after the bisulfide treatment?
That's the wrong enzyme. Use Taq, not a Taq + proofreading enzyme. NEB makes a Taq master mix, but so does every other company. Use one. Unconverted DNA will be optionally methylated at CG sites. The sequence of the primers should exactly match the genomic DNA sequence. There may be many other locations in the genome that also match the primer, which is OK unless both the forward and reverse match within a short distance at several places.
You should definitely concentrate on getting your unconverted PCR to work and ignore conversion for now. I would stop using the word "unmethylated" to describe the primers ampifying converted DNA. While it is accurate, it is non-standard and likely to confuse you (!).
picog is not a concentration, but an amount. And no one writes it that way. It is pg. No one writes microliters as microl. It is ul.
nhy_t on Tue Feb 17 11:11:34 2015 said:
Hi all!
I am trying to design MSP primers for human BCL11A gene based on the DNA sequences from the CpG Islands - 115 & 106 prior the promoter region and 105 towards the end of the gene.
I have attached the Excel file with all the primer sequences for these regions. The graphic representation from the result aligned the primer with input sequences and I double checked the parameters of the primers. However, I couldn't see any bands after running the PCR. The PCR conditions that I used are:
Mixture: 1microl primer, 10microl master mix, 7microl deionised water and 2microl genomic DNA
PCR:
5 mins at 95oC
30sec at 95oC
30sec at 55oC (chosen based on the primer pair with the lowest average Tm)
30sec at 68oC
(Repeat step 2-4 for 40 cycles)
Another question I have is when you are preparing for PCR, do you mix both unmethylated and methylated primer pairs in the same sample mixture but separately?
I am clueless why the PCR's not working. Please help!!!!!!! And thanks a bunch!
Hi,
My advice to you is to reconsider the primer design. I have checked some of the primer pairs from the table. It is very likely that these primers are not specific enough to differentiate between native and modified DNA sequence.
phage434 on Tue Feb 17 13:20:15 2015 said:
The primer pairs are run separately. Are you able to amplify the unconverted DNA with your methylated primers? You need to get that working before even thinking about working with converted DNA. The volumes you give in your PCR setup tell little without knowing the concentrations. What master mix are you using? I'd recommend a Taq based one rather than an expensive high fidelity one. High fidelity ones won't amplify uracil containing templates (which your converted DNA will contain), and Taq is more forgiving in many ways. Have you considered nested or semi-nested PCR?
Hi Phage434,
Regarding to this, you mentioned about amplify the unconverted gDNA with the pair of methylated-specific primers, is it possible to get it works? How if the methylated-specific primers covered the 'C' nucleotide that is not next to 'G'. In this case, it will be converted regardless it's methylated or not right? Will it be possible to amplify the unconverted gDNA in this case?