hello everyone. I need to design primers for full length gene amplification of Gossipium hirsutum followed by transformation in local cotton variety. the sequences are retrieved from NCBI (XM_041098083.1, XM_041116887.1 and have many more like these).
How would I know which gene is already full length, any indication
or pointer that will help to find.
Please guide do I need to start designing primer from the first ATG to the last stop codon in sequence. Or I need to design primers from the start codon of my desired protein present in the sequence.
Taking XM_041098083.1 as an example, you can search for it in the NCBI Gene database. Then scroll down to the NCBI Reference Sequences (RefSeq) section. There, you can click on FASTA to see the genomic DNA sequence. You can then copy and paste this into SnapGene Viewer or other molecular biology software. In SnapGene Viewer, you can click on the "Show translations" button and choose Top 3 Frames. Going back to the NCBI Gene result, you can scroll back to the NCBI Reference Sequences (RefSeq) section and click on XM_041098083.1. Scrolling down to CDS will give you the translated amino acid sequence. Note that it starts with MMA. You can use Ctrl F to search for MMA in SnapGene Viewer. (Choose Find Protein Sequence. Find DNA Sequence is the default.) This will find the beginning of your amino acid sequence, but not the end because there are multiple exons. You can continue to search for all of the exons in a similar manner, making sure that your search query only returns 1 match every time (i.e. use more than three amino acids as needed). The bottom line is that you'll want your forward primer to be 5' of the first exon and your reverse primer to be 3' of the last exon.
Thank you so much @Jeremy for such detailed answer.. it helped me a lot..
I would be grateful if you could answer my first question as well.
A full length gene have a stretch of poly A tail at 3 prime end and GTP cap at 5 prime end. A poly A tail can be easily seen at the 3 prime end but how will I know that 5 prime end has a GTP cap as well.?
You could use NCBI's primer design tool : https://www.ncbi.nlm.nih.gov/tools/primer-blast/ if you have a small number of these. You simply need to paste the accession numbers in the search.