I need to develop an assay to rapidly genotype 7 polymorphisms on 7 different genes, based on a primer extension either with labelled nucleotides or using size separation with Bioanalyzer. Has anyone any experience with multiplex SNP genotyping and if so what is the optimal technique? thanks!
If the polymorphisms have to be detected by primer-extension, the sequences of the polymorphism-specific primers (let's call then "foreward primers") are quite fixed. You would just need to find compatible reverse primers. To my opinion, the simple "empirical way" will give quickly acceptable results: order some possible reverse primers per polymorphism and test all possible combinations in multiplex reactions under standard conditions.Select the combination that works best.
Some software: MPprimer, PrimerPlex, FastPCR, MultiPrimer
use this if it is already known region for established organisms:
http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do
may I ask which equipment you have for the multiplex ( some times companies they do provide such softwares, even for free)
Sequenome has a software for multiplexing up to 15 SNPs. http://www.bmgc.umn.edu/prod/groups/ahc/@pub/@ahc/documents/asset/ahc_30831.pdf
- Badges
- Science method