I need to build a multiple sequence alignment for my project, as I'm designing a PCR assay. I'm confused as to whether I should build the alignment using my PCR product or with the sequence I used to obtain the product from? I feel like using the product is the better option as that is what is going to be amplified and should be checked against other species and strains, but I would like confirmation before I go ahead and put that in my paper. Thank you!
You're going to need to clarify what you are doing. If you already have quality information on the DNA sequence for all of your species/strains then you don't need to do any PCR.
If you are wanting to use the existing DNA information to design new PCR primers to then try and get good sequence from more species/strains/individuals, then you look for the most conserved regions in the alignment of your known samples & put your primers in those regions.
Depending on your region of interest & species/strains of interest, there might be published primers to try.
Good luck!
Katie A S Burnette I've already designed and used the primers as my project is designing E. coli primers to use in a clinical setting for LMICs. My supervisor wants me to include a sequence alignment in my results to show they work in silico (they were confirmed specific when I used them). I ask because I wasn't sure if i needed to use my 215bp product in the alignment sequence or to use the full sequence of the gene I designed the primers from. If you need any more information let me know, thank you!
Dear William,
First align your amplified 215bp products and remove unnecessary bases which make messy bands (according to their intensity of the sequencing garphs).
Then you have to confirm, is this your interested gene region....
*For better interpretation align the gene region you used to design primers with the above aligned sequences....
Use the mega software to align sequences.(sequences should be in the fasta format)
Lahiru Madushan P. Gamage Thank you so much! I've run the sequence through BLAST to get homologous sequences to put in the alignment. This is just to show on my paper that the primers would work in silico before they were ordered but we already did all that and I forgot to do an alignment.
Multiple sequence alignment will give you the global alignment not the local alignment. So you can use the full gene sequence (sequence which you used to generate your PCR product) and align with homologous species. From the global alignment you will get to know all the conserved regions(including your pcr product) between your query and the similar strain or species.
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