I'm having a lot of stop codon in my sequence. Even though i delete it one by one, but still a lot, so i think there's no use to keep on deleting it over and over. it will make my sequence become shorter. My labmates said maybe the problem is from my primer, im using universal primer of coi. So, is it because im using the primer or is there any problem? Thank you.
Check the obtained sequence if it makes sense of your expected gene. If it does, design a pair of sequence specific primers based on the known gene sequence and re-do your gene amplification.
Hi Amelia Kim,
did you check your obtained sequence in terms of all six possibilities sequence reading? If all of the possibilities give the same results (many stop codon), then you should design another primer based on the expected gene and do another round of amplification
You might be reading the sequence in a wrong frame. Check your sequence by subjecting it to the NCBI ORF Finder program (See the link below). It will translate your nucleotide sequence in all the 6 possible frames, and you can look for a meaningful coding frame.
Best wishes
SD
http://www.ncbi.nlm.nih.gov/gorf/gorf.html
Hi Amelia,
Normally primers bind in the first or second position of a codon because they are more conservative than the third one (for the characteristics of the genetic code). So, if you try to translate to aminoacids with a wrong frame obviously is easy to find stop codons. To avoid this, check the position of your primers in the gene, probably you have to delete the first or the two first positions of your alignment. Another (and easy) way is check the total length of your alignment, if the number is not a multiple of 3 you have to review your alignment.
Best wishes
Dear Amelia, in COI sequences you will never get stop codon, its just a matter of frame shift. As suggested by Fen and Sibnarayan, you need to check codon positions. You can simply test by giving one space at beginning and try to encode proteins and find for stop codon if still you will find, again add one space and repeat steps, I am sure you will get rid of stop codons. Its typically sequence alignment issue, no nee to test for primers or else. Good luck.
You may have a pseudogene. We have experienced this when trying to clone cDNA.
Dear Vijay, I suspect on which aligner program you are testing your sequence and which reference you selected. It largly depend on these two criteria. Please use any standrad reference and then compare, dont put everything at codon frame 1. I believe, you will do it.
Dear Dr. Ayyagari, I wonder why for exactly the same sequence this bioinformatics-sms you have suggested gave me not the same answer as the one (NCBI) suggested by Dr. Datta , please? Thanks.
Ok, l will recheck Dr. Ayyagari. Anyway, could you please lend me your hand on what application to be used to find out the name of the gene resulted from the sequence. Thanks.
Dear DRs. Alzoughool & Ayyagari, yes you were correct, the two links give me the same ORFs. Thank you.
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