Hi all,
Recently, while PCR-amplifying a chloroplast DNA (cpDNA) spacer region in a tree species, I observed 2 samples (out of a population of 15) that showed double bands (for a single locus) on the gel after electrophoresis, with one of the bands having the same size as the amplicon of other samples that showed only 1 band. At first, I thought it was contamination, but it did not seem likely as the other samples (extracted in the same process) were fine. Anyway I still tried re-extracting the gDNA for those 2 samples and it still yielded the same result. I then tried to PCR-amplify 4 other cpDNA loci (including those for barcoding purposes) in those 2 samples and 1 negative control, but no double bands were found in those loci. Does anyone have any experience in this? What could it be? Some people told me that it is due to a duplication of a certain gene fragment within the amplified region. But is there a way to prove the existence of the duplication, or otherwise? I would really appreciate it if someone could enlighten me on this, or point me to a related article. Thank you!
Hi Wei,
Sounds like your friend may be right in that its a duplication of the gene, other possibilities are its another gene in those samples that have similar primer binding sites. DNA-DNA hybridization (southern blots) is one way to check for the number of genes/copy number you may have. I have not tried this with reference genes, or non-genic regions like you seem to be using. The fastest method to see what the other band is would be to purify that amplicon and get it sequenced. This is relatively easy and for just a couple of sample (less than 5) many sequencing facilities will offer free promotional deals. Take a look at Eurofin for example (http://www.operon.com/default.aspx). Once you have the it sequenced you could BLAST it at NCBI. Contamination may still be a possibility, mistletoe comes to mind.
Hi Paul,
Thank you very much for your reply. I sent my PCR products for sequencing right after posting the question and I just got the sequences today. Apparently, after BLASTing, one band resembles cpDNA sequences of the species I am working on, while the other is highly similar to that of a lichen's cpDNA sequence. I did not know that primers for a universal cpDNA marker in plants could also amplify lichen DNA so I did not suspect contamination in the starting material. A little disappointing, but well, this is science :-) Thank you again!
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