Taqman probe design using FISH probe (need help)

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Post by protoemics » Wed Feb 04, 2015 8:11 am

Although they are both called "probes", FISH and Taqman probes have different characteristics. Probes for FISH need to be at least 1 kb long, to hybridize and "highlight" the target chromosomal DNA (metaphase chromosomes or interphase nuclei). Hydrolysis probes (such as Taqman) are very short and used in combination with other two oligonucleotides. the size of the amplicon is very small, usually < 200 bp. here, the hydrolysis probe has an annealing temperature such that it will remain hybridized at PCR extension temperature (but not so high as a fish probe) and will be hydrolysed by Taq DNA polymerase at every replication, thus releasing the fluorescence (quenched in intact probe) that will be measured to quantify the starting level of template.
Therefore, you can design primers and probes so that they will fall within the sequence recognized by the FISH probe.
you can easily find instructions for designing good quality primers for qPCR.
Good luck!

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