Dear all, I want to find a specific primer for genus Sphingobium, could you give me some idea on it? Thank you so much~
Hello Yan Li
You have to consider first that the genus Sphingobium (NCBI:txid165695) contains several species, according to the GenBank database, are available 37 complete genomes and more than 24,600 nucleotide sequences. (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=165695&lvl=3&lin=f&keep=1&srchmode=1&unlock)
Therefore, considering that you want to design primers that detect almost all species of the genus, you should use all the complete and annotated genomes available from all species, align them (using MAUVE for example), look for conserved genes (preferably housekeeping genes like rpoB, infB, RNA16S, etc), then you can extract the alignment from the interested region. Once you find conserved regions, design primers and compare them with the nucleotide database that exists for that target region. Once analyzed in silico your candidate primers, you can proceed to make tests for specificity, sensitivity, etc.
In short, you should perform an in silico bioinformatic analysis first in which you should look for a conserved target region in the genus for the design of the primers. Then check with the rest of the database. And finally, do the practise tests ( in vivo)
If my answer has been useful, please recommend it.
:) :)
Good luck!
Hello again Yan Li ,
Now I understand that you want to design a PCR to detect strains from the species Sphingobium phenoxybenzoativorans. In that case, the housekeeping gen 16S rRNA selection is not a bad idea. But in my opinion, also look for the more abundant nucleotides from the database could be useful. Remember that other housekeeping genes can be used with the same objective. Good luck !! :)
Of course Yan Li ,
regarding the software, you can use a lot to make alignments. Mega software (www.megasoftware.net) is free and useful for small alignments, in my opinion, < 10Kbp. And also is very useful for phylogenetic analyses. For big alignments (or example, whole genomes alignments from bacteria f), MUSCLE is very useful too. And have big advantages if the genome alignments are performed with the annotated genomes because you can find the ORFs (or genes...;) ) in a searcher includes in the software. For example, you can search 16S RNA gene, rpoB, infB... etc (https://pubmed.ncbi.nlm.nih.gov/15034147/)
Others free software with an interface is Unipro UGENE (http://ugene.net/) VERY USEFUL because you can integrate plugins at the software... like alignments methods (MUSCLE, Clustal W, etc...). But if you have money, my recommendation is to use Genious software, that includes almost every package useful for your essay (https://www.geneious.com/).
Regarding the database: NCBI is the best and more used (Nucleotide and genomes).
https://www.ncbi.nlm.nih.gov/nuccore ;
https://www.ncbi.nlm.nih.gov/genome/
To find the conserved region you have to think first about
What genomes in the bacterium are more conserved? answering this question you can find putative genes useful for your PCR design.
Good luck!!
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