Help: multiplex SNP markers

Dear Colleagues,

Does anyone have experience with multiplex SNP markers?

We are trying to multiplex 15 SNP markers in 3 sets using SNaPshot multiplex ready kit. In order to improve multiplex optimization, we added poly (dA) tail with 4 bases differences and then ran them in singleplex system to check the length of each product; however, it didn’t work as expected because some markers produced overlap sizes. When we increased or reduced the number of nucleotides, inconsistent size resulted. In some primers, reducing four nucleotides did not produce any difference in size and when we added a number of nucleotides, instead of longer size, shorter size resulted.

1. Does the kind of tail added in the 5’ primer sequence influence the result (although the manual protocol of SNaPshot mentions that generally poly (dT), poly (dA), poly (dC) and poly(dGACT) tails won’t affect the signal patterns, probably any consideration for choosing which 5’ non homologous tail will work best.
2. How many number of nucleotide difference will significantly affect the size?
3. Are there any general tips for multiplexing SNP marker, such as design of primer, Tm etc?

Your suggestions concerning the matter above will be appreciated.

Thank you
Vivi Yuskianti

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