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Primer designing

First, designing the Primers for the Allele-Specific PCR is necessary to obtain the sequence downstream and upstream of the targeted variant. We used the Ensembl database to load the following sequence. In the database settings, we selected to present only the variants with MAF > 0.01%.

Then, we copied the FASTA format of the sequence, and we used it in the Primer3 tool to design the primers for the variant. Considering that the forward primer of the AS-PCR has to contain as the last base of the 3’ end the targeted variant, we choose the sequence and the length of the two forward primers. The reverse primer was designed by the Primer3 tool.

After we checked the Tm temperatures and the lengths of all three primers and the DNA fragment’s product size, we tested the primers’ combinations through the UCSC’s in silico PCR. We saw that any of the two combinations Fw-R and Fm-R, produce a unique PCR product.

We also tested the primers through a BLAST search to determine if they have any other hits apart from the targeted region. The search showed that the Fw and Fm primer has three hits in different chromosomes. The reverse primer has two hits in different chromosomes.

The folding and hybridization of the primers and the PCR product were also tested with the DinaMelt, an online tool predicting RNA and DNA’s secondary structure, mainly using thermodynamic methods.

The DinaMelt results were the following:

DinaMelt Two-state Folding Results for the PCR product.

DinaMelt Two-state Folding Results for the Forward primer for the ancestral allele.

DinaMelt Two-state Folding Results for the Forward primer for the alternative allele.

DinaMelt Two-state Melting hybridization for the Forward primer for the ancestral allele and Reverse.

DinaMelt Two-state Melting hybridization for the Forward primer for the alternative allele and Reverse.

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Primer designing

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