Part:BBa_K3464002:Design
Reverse Primer for the detection of the rs4149056
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design
For the designing of the Reverse Primer was necessary to obtain the sequence downstream and upstream of the targeted variant. We used the Ensembl database to load the sequence. In the database settings, we selected to present only the variants with MAF > 0.01% to avoid the primer locating upon another common variant.
Then, we copied the FASTA format of the sequence, and we used it in the Primer3 tool to design the primers for the variant. The reverse primer was designed by the Primer3 tool.
After we checked the Tm temperatures and the lengths of all three primers, and the product size of the DNA fragment, we tested the combinations of the primers through the UCSC’s in silico PCR. We saw that the combinations Fm-R and Fw-R, produce a unique PCR product.
We also tested the primer through a BLAST search to determine if it has any other hits apart from the targeted region. The search showed that the Reverse primer has 2 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 by using thermodynamic methods.
DinaMelt Two-state Folding Results for the Reverse primer.