When I make NCBI blast (nucleotide sequence) and write rats in the organism, I find the ident and query cover 100% for the gene that I made primer design for it, but also I find ident 100% and query value (less than 100%) for other genes, what does this mean?
Dear Mai,
I suppose you used as query the amplified fragment not the PCR primer sequences.
The result you obtain means that highly similar sequences exist in the genome. They may represent different genes arising from duplications and divergence (eg. different NOTCH genes) or pseudogenes.
Ok that means your primers may anneal with the similar sequences. This should not be a problem unless the identity is not 100% and if the mismatches are at the 3' of the primers, to avoid the non specific amplification you can tune primer concentration and/or annealing temperature. Anyway I would suggest you to perform an in-silico PCR with the UCSC genome browser tool (or anyother you feel more confident). If you get only one predicted product go on with your experiments
I used UCSC In_Silico PCR, but the size of some products is very large than that I previously calculated using primer3 and amplifx programs and this problem appeared also with the primers that are used in previous researches
Sometimes presence of repeated elements in introns (eg. transposon) generate errors in automatic genome assemblies. I would suggest you to perform the PCR on gDNA and sequencing of the fragment, then submit it to GenBank as alternative sequence of the region you are working on
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