Part:BBa_K3464001:Design
Forward Primer for the detection of alternative allele 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 Forward Primer for the Alternative allele 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. Taking into consideration 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 primer. 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 combination Fm-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 Fm primer has three different 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 by using thermodynamic methods.
DinaMelt Two-state Folding Results for the Forward primer for the alternative allele.
DinaMelt Two-state Melting hybridization for the Forward primer for the alternative allele and Reverse.