We have been working on Banana streak virus. The PCR product which was sent for sequencing, after clonning, gives two different sequences from two different colonies. Similiarly, It was also observed that Musa cv. Pisang Klutuk Wulung (PKW) and OL complete genome has more than one regions with more than 90-95 query coverage? How can we explain such results?
Upon reading your question, my first instinct is that you may be seeing that your primers are binding not just to where you expect, but also binding with one or more mismatches to another genomic region. It sounds like some troubleshooting should be done so that you can eliminate mis-priming as a confounding factor.
A few simple experiments: 1) raise your hybridization temperature by a few degrees, since higher temperature will disrupt imperfect pairing in 'mis-priming' events. This will normally reduce yield of the expected target, so you might add more cycles to offset. 2) move both primers in or out, so that they have difference sequences, and perhaps you will eliminate any 'mis-priming' events. 3) nested PCR: amplifying with a second set of primers using your first amplicon as the template. In this case, 4 primers give better target sequence specificity than only 2 primers.
Hi Rupert,
it seems that your favorite virus share most of its sequence with other viruses. but this is not surprising. Searching in the NCBI website you'll find 3544 viruses with their sequence. I suggest you to blast the waited sequence of your amplicon on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&SHOW_DEFAULTS=on&BLAST_SPEC=MicrobialGenomes), see what match with your virus, get all relevant sequences and redesign new oligos in the most mismatching region.
fred
Fred is right...
Different genomic regions from the same isolate, mean that the used primers are not specific enough.
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