Hello everybody,
I need to barcode some mites amplifying by PCR a stretch of their mitochondrial Cytochrome Oxidase I (COX I). But I don't have its exact sequence, as this is the question the obtained PCR product is expected to answer after sending this PCR fragment for Sanger conventional dideoxy DNA sequencing.
I've already used before a pair with degenerate primers with one of them containing a stretch of 6 consecutive inosines and it (fearfully!!!) worked out just fine, as published here: Article Morphological and molecular identification of ticks infestin...
The calculated degeneracy of such primer is an elevated number of 4096 (4x4x4x4x4x4) and it worked out fine in spite of it.
But now I need to amplify this COI fragment without knowledge of its exact sequence. I've already aligned the closest available species with publicly available sequences in order to find the most probable degenerate primer able to amplify any of them (at least in theory).
I've been aligning these sequences searching for a common stretch among them with the lowest possible degeneracy that's well below 4096, actually equal or below 300.
At last here comes my question, which number would be roughly the top possible for a degenerate primer to amplify a single DNA product? 500? 1000?
Is it much different for these bases to be scattered along the DNA primer instead of a single long consecutive stretch of degenerate bases (such as the 6 consecutive inosines I've already used successfully)?
Thank you all in advances for the attention, and also for the patience needed to read such long considerations.
Another relevant information for those interested in this question: I know it's possible to amplify the desired DNA fragment with a nested PCR with TWO PAIRS of degenerated primers instead of just one pair. Since custom oligos have becoming increasingly cheaper this is already an available solution, but I wonder if this is the only possible way to solve my problem or if somebody else has another insight into it.
Try OMEGA primers where you use around 10 bases of homology with another region down stream and insert a loop of 18-24 bases. The two homologous or minimal degeneracies stretches are additive because their proximity makes each concentration of the two homologous stretches infinite. A powerful technique. I will happily help you at: [email protected]
Good luck,
Alex Ryncarz PhD
Ruslan, even after installing a trusted certificate everytime I try to run the software a small window blinks briefly (probably some cmd command prompt) and nothing else happens.
Thank you Alex, but I wonder how to design such primers with limited similarity/identity among several sequences. In this case I suppose they are inside the loop of the OMEGA primers. But what about the 2 annealing segments without enough identity/similarity in order to bind to the desired gDNA? Are these primers designed by a specific software or is it possible to design them by hand? What's the required identity/similarity to template in each of the 3 segments of the OMEGA primer?
Will get you a publication. Don't let me forget.
Work done with N Kurn and Edwin Ullman.
Finally, after so much time, I was able to run the program, with the instructions to introduce exceptions into the JAVA console. Nice program, good job!
People still interested in this question perhaps may find useful an Excel spreadsheet I've designed with an automatic primer degeneracy calculator function in one of the spreadsheet columns (Column "D"). It's freely available for download in my "data" section (hyperlink cited below) and this is how it works: one may copy & paste their primer names and sequences in the first 2 A & B columns, respectively. 3rd column is reserved for degeneracy calculation and 4th column gives primer's degeneracy number automatically. Column C lists all nucleotide symbols supported according tho the IUPAC nomenclature, including inosine as well as degenerated base pairs. The spreadsheet calculation is able to deal with up to 10 thousand primers for those working with massive amounts of data. People interested in extending this capability may use the Excel spreadsheet's autocomplete function in the lower right corner of the selected cells.
Method Oligonucleotide/Primer Degeneracy Automatic Calculator
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