random priming in FISH (fluorescence in situ hybridisation)
NoReply at email.com
Wed Dec 28 11:28:46 EST 2005
In article <1135779583.854902.234870 at f14g2000cwb.googlegroups.com>,
doublemind at gmail.com wrote:
> I'm currently writing a coursework about FISH for school and have a
> question about random priming as a means for probe labelling.
> As far as I understand, for random priming, the template DNA (i.e.
> probe DNA in FISH) is mixed with short DNA-sequences (=random primers)
> which have every possible base combination, so that almost every bit of
> template DNA is covered by these primers. Then, DNA-polymerase is
> added, which attaches fluorescence marked single nucleotides to the
> ends of the random primers. These primers are now the labelled probe.
> This is how I got it from my sources, but it cannot be right. If the
> probe was made of random primers, and these random primers had any
> possible base combination, the probe would not bind specifically to
> some place on the target DNA but would be scattered everywhere because
> the random primers are so variable.
> I hope I described my problem understandably,
> thanx for your answers in advance,
When the random primers have bound, they (as their name suggests) prime
DNA synthesis by the DNA polymerase. The polymerase then copies the
template for several hundred bases (or more) incorporating the
fluorescently tagged bases as it goes, thus making your specific probe.
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