too many hits on Blast for primers / microsatellite - (Apr/24/2007 )
Hi there!
I was looking for microsatellite markers for genotyping mice. We have Jackson mice so I went to their webpage and simply downloaded some primer sets. But my problem is that when I BLASTed them, I found that both the forward and reverse primers got hits from almost (or completely) every chromosomes! Now how can I trust the product size they published in papers? Will I really get just one band? I don't want to check out all the 30-40 possibilities for each of my 4 primerpairs to find out if they don't have any other product which is in size will be near to the one I expect... Does anyone have any experience with it...?
Hi!
I guess it depends on a lot of things.... You say you get many hits; If one primer gives you e.g. 30 blast hits, it is extremely unlikely that the wrong hits is in vicinity of a sequence identical to the reverse primers, so it might work all right. Yet, multiplexing, if that is what you´re doing, can be tricky even though its simple in theory. I guess multiplexing with a primer mix that binds "every where" might be problematic. How about mailing the corresponding author of the original paper?
Cheers
I was looking for microsatellite markers for genotyping mice. We have Jackson mice so I went to their webpage and simply downloaded some primer sets. But my problem is that when I BLASTed them, I found that both the forward and reverse primers got hits from almost (or completely) every chromosomes! Now how can I trust the product size they published in papers? Will I really get just one band? I don't want to check out all the 30-40 possibilities for each of my 4 primerpairs to find out if they don't have any other product which is in size will be near to the one I expect... Does anyone have any experience with it...?
Hi Chimp,
thank you for the answer. In fact, I just want to genotype my mice to know from which parents they were derived from. I wrote e-mail to some authors in this topic but the replied I can find all these informations in tha Jackson Lab homepage. And I foun sequences, it's ok. But when I blast them... but ok, I've ordered the primers, I'll see what will happen.
cheers
J.
I hope it works out!
thank you for the answer. In fact, I just want to genotype my mice to know from which parents they were derived from. I wrote e-mail to some authors in this topic but the replied I can find all these informations in tha Jackson Lab homepage. And I foun sequences, it's ok. But when I blast them... but ok, I've ordered the primers, I'll see what will happen.
cheers
J.
Just check the E-value.
A quote from the NCBI/BLAST FAQs:
Q: What is the Expect (E) value?
The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences. For example, an E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance. This means that the lower the E-value, or the closer it is to "0" the more "significant" the match is. However, keep in mind that searches with short sequences, can be virtually indentical and have relatively high EValue. This is because the calculation of the E-value also takes into account the length of the Query sequence. This is because shorter sequences have a high probability of occuring in the database purely by chance. For more details please see the calculations in the BLAST Course.
The Expect value can also be used as a convenient way to create a significance threshold for reporting results. You can change the Expect value threshold on most main BLAST search pages. When the Expect value is increased from the default value of 10, a larger list with more low-scoring hits can be reported.