I am currently using genetyx and clustalw for alignment.
Hi, I guess you are asking for DESIGNING DEGENERATE PRIMERS. Find the attached file containing basic of it, which may help you. Good Luck
We are using Mega for clustalw alignment and then make degenerate primers. If you find a good match for your all sequences it would be enough.
You just align all the sequences using MEGA and save it as fasta format.
After that you will import it in HYDEN software for primer designing. The generated primer sets becoms a good primer for amplifying all the sequences. You can also checkout the fidelity of primer using insilico tools.
Some programs that align DNA sequences include an option to mark matching identities with a dot to help identify portions of DNA that are conserved between aligned sequences. I tend to use BioEdit for this purpose, but there may be better programs with similar functions. Once you identify which areas of the gene are conserved between your samples, it is easier to identify good sequences for primer creation and determine which nucleotides may need to be degenerate.
choose two region most similar in alignment and design forward and reverse primer using those sequences
Thank you everyone. In the end, I aligned using clustalw and used used primer 3 to get appropriate primers since qPCR has specifications. I adjusted the region for primer search to the regions that have the most similar alignments and from there I manually chose and got 2 pairs of primers that could amplify the whole 62 sequences. Do you think this is okay?
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