Need the suggestion and help in Tetra ARMS-PCR standardization ... Thanks in advance.
1.What are the optimal/best conditions and reagent concentration?
2.Which one we should standardize first? Tm or denaturation/extension---Time or Temp.
3.IF we dont have positive sample how we go ahead?
4.IF alternate alleles are more than one and we are able to design the primers for it how best T-ARMS works?
We designed our primer using https://snp.biotech.edu.lk/
Problems we faced
1. We are able to get Outer and wild allele bands but not the variant allele band
2. In negative we are getting primer dimer along with another 50bp dimer/band With One SNP
3. Non specific bands
We Tried with
1. Gradient PCR for tm
2. Different primer concentration Outer Vs Inner Primer (1:10, 1:20)
3.We also tryed with increasing the variant allele primer conc.
4. Used different time and temperatures
Tm are
SNP1 OF-72* OR-74* IF-73* IR-73*; outer band 275bp, Wild allele 179bp and Variant allele 152bp
SNP2 OF-69* OR-69* IF-69* IR-69*; outer band 361bp, Wild allele 218bp and Variant allele 195bp
We used PCR conditions
94/95*c-----2 to 3 min.
95*c-----40 to 60 sec.------/
55 to 68*c-----20 to 30 sec / 20-35 cycles
72*c-----40 to 60 sec-------/
72*c-----3/5/10 min
Reaction concentrations
OF---- 10 to 20 pm
OR---- 10 to 20 pm
IF---- 10 to 100 pm
OF---- 10 to 100 pm
master mix (2X) 6-8 micro lit. Emarold master mix (takara)
Thank you..
At least if you are getting the outer primers to work than you have a lot of amplified material to work with since this amplimer can be used heavily diluted in for the inner allele primers while you get the temperatures sorted out. In answer to your Questions I would not change any concentrations except perhaps Mg if needed as all of the pcr reagents are in huge excess and a mixture of primers will not make much difference.
I would not bother with extension times as all these enzymes work very quickly ( well above 100bases/second) but the temperature profile is very important in ARMS so running gradients of the annealing temperatures against the mutant and normal dna sample is very important so that you can see what temperature will work best in a primer mixture
Primer dimer is normal in no template controls when the enzyme has nothing to do but does try to do something. If the real samples are working well the PD is usually minimal but if it is a problem then use a hot start enzyme or half the amount of primer ( it will still be in huge excess).
Non specific bands can be dealt with when you are getting all primer sets to amplify . If they are distant in size they can be ignored or you can try using DMSO added to the mix later on when things are working to tidy up the bands although this may change the annealing temperature.
I see that you are using Emerald Taq. You should read the instructions that come with the Taq.
It has a high thermal stability and recommended denaturation is 98c ( I think).
The question of what to do if you have no positive control is harder. Contact labs that work with positive samples and beg some of theirs. Heterozygotes can be just a mixture of homozygote and normal dna. Buy a commercial gene block of the mutant allele. Clone the normal allele and use site directed mutagenesis to get a mutant sequence plasmid. Sequence lots of likely clinical samples which may be mutated until you find one perhaps
Please read these papers, it may help.
Article Genotyping single nucleotide polymorphisms in barley by tetr...
Article An efficient procedure for genotyping single nucleotide polymorphisms
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