I have my pcr produkt with forward and reverse primer. We sent it to sequence from both sides and then i got different forward seqence and different reverse. When i did my philogeny analysis it shows that reverse sequence is pseudogen. How is it possible that this reverse primer is alinging with pseudogen and forward with my gene of interest?
Primers are short sequences that are designed to pair to specific parts of a gene; however, sometimes there is not 100% match and there is a chance that the primer finds a similar region in other part of the genome and attaches to it.
I can suggest you to check literature on the taxa and marker you are sequencing and see if there are other primers available. Other option is that you design a more specific primer for your work.
the sequence of a pseudogene is often very similar to the cdna sequence ( exons only) of the gene so if you designed the forward primer in intronic sequence and the reverse primer in an exon then the forward sequence will contain intron and will match the gene sequence and intron so will not match the pseudogene sequence completely but the reverse primer being in the exon or just outside an exon but producing sequence which is completely coding sequence ( the primer and nearest 40 bases do not sequence in Sanger sequencing) so the sequenced part of the amplimer will be a very good match for the pseudogene sequence because there is no intron sequence to force the alignment with the whole gene
One more taught: My product is mitochondrial so there are no exons and introns although bigger problem is (not problem but we just don´t understand mechanism how is that possible) that it is a short product and i get exact same length product from both sides with primers sequenced. The question is how it is possible that reverse amplifies pseudogen and forward primer amplifies gene of interest, when i sent the same primers with which i did PCR in the first place?
I think that introns can exist in mitochondrial dna but it has to be self splicing as mitochondria lack splicing mechanisms.
Is the pseudogene on genomic dna or mitochondrial dna ?, If on the mitochondrial dna then this exists in multiple copies in each mitochondrion. If the sequences are very similar but the pseudogene has accumulated mutations mainly at the reverse primer end of your amplimer then you may have amplified both gene and pseudogene but there were more copies of the pseudogene then the common sequence end might analyse as gene but the sequenced changes in the other end would be more like pseudogene. Are the dna sequences up and downstream of each target different so that you can design gene specific primers which will not amplify pseudogene?
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