Hi all,
I'm trying to perform ARMS PCR to see if certain cell lines that I'm interested in show heterozygosity for SNP rs2736098 (in exon 2 of TERT gene)
This is my first time doing ARMS PCR and practically don't have any idea how to design a primer for this.. I understand the theory but I'm worried that Primer design may be different from standard PCR.
Input from experts in this aspect would be greatly appreciated !
primer design is a bit different. The technique relies on a single base mismatch at the 3' end of the primer not annealing and so there is no pcr product. This is true for strong interactions but if the mismatched bases interact weakly it may be necessary to introduce a second deliberate mismatch next to the 3' end mismatch in your primer. This can only really be detected by experimental means . The paragraph below lifted from a google search indicates which mismatches may need more work. If it does not work you could consider artificially created restriction sit pcr ( google dcaps) which is very specific and has a web site to design your primer
As a general rule for ARMS primer design, if the 3' terminal mismatch is a weak one, a strong secondary mismatch is engineered. If it is a strong one, a weak secondary mismatch is introduced. Try putting the mismatch at the second nucleotide in the first instance and test the primer for specificity and generation of product. The position of the mismatch can be altered if the primer does not work, or the strength of the mismatch increased if non-specific bands are observed. According to Little in Current Protocols in Human Genetics (28), the strength of mismatch pairings are; maximum, GA, CT, TT; strong, CC; medium, AA, GG; Weak, CA, GT; none, AT, GC.
I would like to share some more useful links on ARMS-PCR
https://www.researchgate.net/publication/272076871_Modified_Tetra-Primer_ARMS_PCR_as_a_Single-Nucleotide_Polymorphism_Genotyping_Tool
Designing tool : http://primer1.soton.ac.uk/primer1.html
Article Modified Tetra-Primer ARMS PCR as a Single-Nucleotide Polymo...
The following web-based software can help you on the design.
Wasp
http://bioinfo.biotec.or.th/WASP
It works pretty well. Just enter the SNP code rs2736098. If the software does not generate any primer set, try to change the design parameters.
All the best
You can design your ARMS PCR primers by using the following link: http://primer1.soton.ac.uk/primer1.html
SOS: Looking to PCR and then sequence alternatively spliced PCR amplicons.
Sorry to hijack this question, but I have a similar-ish question. I have attached an image to illustrate the problem I'm coming across.
In simple terms, I have found a mutation in a gene called ATP11A. This mutation is a substitution from a G to an A at the end of an exon. According to standard splicing rules, it is very likely that this will cause alternative splicing. I decided to do reverse transcription PCR with primers that flanked my splice site mutation. There is evidence of alternative splicing because I see multiple PCR amplicons that are specific to the affected individuals that are heterozygous for a suspected splicing mutation.
This is where I am running into issues. Human ATP11A has 19 different ensemble isoforms, which is why I am picking up so many amplicons. I wish I could just sequence this, but that isn’t possible with multiple amplicons. I thought I should TOPO TA clone these amplicons, but they are so close together and it makes it very difficult for gel extractions. Now what I’m thinking is that I should try to do an RT-PCR, but use allele specific primers in my PCR master mix. I have never done a PCR that uses allele specific primers and was looking for advice about how to go about doing this? I mostly do touchdown PCRs with primers that are 20bp in length, but I am coming across protocols that are using primers that are 25-30bp long and apparently require a significant amount of optimization. Also, I see some people using forward primers with a mismatch on the 3’ end that is specific to the allele of interest, while other people introduce a mismatch 3bp from the 3’ end of their forward primers to increase specificity. Any recommendations on proven protocols would be greatly appreciated. I am aware of the primer design tool (http://primer1.soton.ac.uk/primer1.html), but I’m wondering how it works. Do I just go with default settings? Or do you change these parameters?
Do I need to do ARMS, using non-specific outer primers and inner primers that are alleles specific? Or could I just do a single PCR with a non-specific reverse primer and an allele specific forward primer? If anyone has any suggestions, it would be greatly appreciated! Does anyone have a validated protocol? Since I don’t know the exact sequence identity of my alternatively spliced amplicons, would it be safe to just assume partial intronic inclusion for primer design?
Thanks for this illustration about T-ARMS PCR design.
I have major issue about Little protocol for deliberate mistach.
Could anyone discuss this protocol with example since I have confused with it? Thanks
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