Our research work involves identification of Ganoderma spp. isolates using ITS region. We purified the PCR product and then sent for sequencing. Prior to PCR amplification, we cultured the fungus on PDA and extracted the mycelia DNAs using conventional method or available kits. However, we are now facing multiple signals problem in the acquired sequencing results. One way to counter this problem is cloning and this work is currently undertaken. Another primers for identification such as transcription elongation factor (tef) etc will also be used in the next PCR amplification.
Nevertheless, we would like to get any suggestion, recommendation or advice regarding this multiple signals problem from your experience and expertise.
Hi Salwa Abdullah Sirajuddin
sequencing issues find often their origins in PCR miss amplification. How did you design your primers? are they specific to your target species? are your samples free from contamination?
the first thing you can try is to test your primers in primer-blast from NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). just put your primers and change the organism, and you'll see what you're supposed to amplify.
fred
Hi Salwa Abdullah Sirajuddin
From my experiences, problems with sequencing have always been either because of inadequate DNA concentration (either after extraction or after PCR) or by using an ineffective PCR DNA polymerase (low processivity or no proof reading).
Since there are a variety of ITS primers, I recommend this article that worked with the Ganoderma genus.
- Jargalmaa et al. (2017), Taxonomic evaluation of selected Ganoderma species and database sequence validation. PeerJ 5:e3596; DOI 10.7717/peerj.3596
Also this one which did not identify single-genus species:
- Abe, C. et al. (2015), Fungi Isolated from Maize (Zea mays L.) Grains and Production of Associated Enzyme Activities. Int. J. Mol. Sci. 2015, 16, 15328-15346; doi:10.3390/ijms160715328
Tef amplification will be useful for comparing with database ITS fragments and further validating your results, but first you have to get the sequencing right.
Hello Frederic Lepretre & Fabiane Cristina dos Santos,
Apologize for the late reply.
Thank you very much for your suggestions and journals recommendation. We really appreciate them.
As for now, we're currently trying to ascertain that our samples are free from contamination as Dr Frederic mentioned above and to test the role of pre- and post-processes from DNA extraction to product purification as Dr Cristina suggested.
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