Hello all,
I am faced with a small problem regarding a set of co-culturing experiments that I am doing at the moment.
I have two Bif strains that are highly similar in sequence (>99.5% sequence similarity) that I would like to co-culture on different carbon sources. I would also like to try each strains' growth and since OD measurements won't do that I was hoping to do qRT-PCR or just qPCR to determine the prevalence of each the strain in the total culture.
Issue is their sequence similarity. Would you happen to know of any scripts/online resources/software that could help me select ideal qPCR amplifying regions that could differentiate between such similar strains?
Cheers!
Dear Ilias, I do not know about exact sequence of Your strains, but in the same case I would align the sequences with any software (vectorNTI, DNAstar, SnapGene or free Ugene). After that you will see not simillar regions. If the sequence is differ enough you can design oligos. If specific region lengts is unsufficient the sequencing is the best way. You also can use DNA nanosensors or non NA-based approaches. May be it helps You a little bit.
Hi,
in fact what John V. Stokes
described is the experimental setup for Probe based SNP detection and should aslo work for your particular setting in case you find sequences which are discriminative. You can further improve the discriminative power by using modified Nucleotides like LNAs.
I have used VectorNTI before to align the sequences (from NCBI) and then make primers on regions that are different. Quite easy software to use, it will just colour every base that's different red and you can scroll through it!
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