primers dont match FASTA gene sequence... 'm confused - (Nov/20/2007 )
I have these three primer pairs. When i BLAST them i get hits on the my gene of interest but when i try to find their sites of attachment on FASTA sequence (which i copied and pasted on a word document)i get a match for only one pair of primers. I am a bit confiused. (i basically want to know where exactly these primers are getting attached to the gene and what is the sequences of the PCR product i'll be getting with them.)
Try this websit, very helpful. Will tell you where your primers bind, product size, Tms, GC content etc.
http://biotools.umassmed.edu/bioapps/primer3_www.cgi
otherwise need a bit more detail to be able to help.
Thank you for your reply. i will certainly take detailed look at the website you recommended.
In the meantime here is a little more detail of my problem. You see when i enter these primer sequences on BLAST, the program shows that the pimers bind in my gene of interest. When i calculate the distance between the two binding sites i get the PCR product of the right size. Now if i were to copy the sequence of this gene and paste on a word document then find sequences matching the primers ( i would of course have to use reverse compliment sequence to one of the primers) I should be able to find them and also locate the PCR product but that doesnot happen.
When you paste "the seqeunce of the gene" where do you copy it from? Blast will search the entire Genbank, and you may be finding sequence outside of the specific gene mentioned. If you then locate that gene, you may miss the matching location if it outside the coding region.
In the meantime here is a little more detail of my problem. You see when i enter these primer sequences on BLAST, the program shows that the pimers bind in my gene of interest. When i calculate the distance between the two binding sites i get the PCR product of the right size. Now if i were to copy the sequence of this gene and paste on a word document then find sequences matching the primers ( i would of course have to use reverse compliment sequence to one of the primers) I should be able to find them and also locate the PCR product but that doesnot happen.
are you using word's search feature? if so, are you putting in the proper string to search (with any spaces, hyphens, capitalization if not agnostic, etc)?
In the meantime here is a little more detail of my problem. You see when i enter these primer sequences on BLAST, the program shows that the pimers bind in my gene of interest. When i calculate the distance between the two binding sites i get the PCR product of the right size. Now if i were to copy the sequence of this gene and paste on a word document then find sequences matching the primers ( i would of course have to use reverse compliment sequence to one of the primers) I should be able to find them and also locate the PCR product but that doesnot happen.
are you using word's search feature? if so, are you putting in the proper string to search (with any spaces, hyphens, capitalization if not agnostic, etc)?
totally agree with mdfenko, word's search feature is not that good for that, had bad experiences myself. in fact, if is not too big a sequence and you know where your primers should match, i'd recommend to do it by eye, bit of a pain but rarely fails !!
Have you tried searching your original sequence AND its reverse complement?
Your primer sequences are 5'->3' right? So if you search only one strand of your sequence you'll only find one of them because the the other one is on the reverse complement.
You can also determine the reverse complement of the primer you do not find and try to find it in your original sequence.
But I agree with the previous posts, word search feature is not very good for this kind of stuff. Try using a sequence analysis software.
are you using word's search feature? if so, are you putting in the proper string to search (with any spaces, hyphens, capitalization if not agnostic, etc)?
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Yes i tried using Words 'search' feature but i was carefull to remove all extra spaces etc so the sequence was continuous. i did find matches for one pair of primers. but not for the other two.
Your primer sequences are 5'->3' right? So if you search only one strand of your sequence you'll only find one of them because the the other one is on the reverse complement.
Yes i did try with the reverse complement sequence. no luck
when i say i "paste the sequences of the gene", i am refering to the sequence that is displayed when i search the gene in the nucletide database on NCBI pubmed. you may be right in that the matching location maybe outside the coding region. {PLease forgive me if sound too ignorant but i am a bit new to molecular biology (medicine is my main field)} do you mean to say that matching location maybe in an intron region and that might not be displayed in the gene sequence displayed by the nucleotide database? Alternatively the matching location maybe outside my gene altogether, maybe in the adjacent gene?