On 3 Oct 1996, David N. Levy wrote:
> What is the limit of in situ DNA hybridization sensitivity? Can one
> detect a single copy gene, and of what length minimum? I assume a
> riboprobe labeled throughout with 32-P or digoxygenin dUTP would be
> most sensitive - correct?
David,
I'm assuming, since you mention single copy gene, that you are asking
about in situ for gene localization on chromosomes (rather than for
transcript localization in cells and tissues). If that's not it, please
ignore the following :)
In situ can certainly detect a single copy gene, limit on length is,
hmmm, depends on the skill and patience of the person trying to map, but
lower limit about a kb of unique sequence. Sometimes 7-800bp, but
difficult to optimize conditions. Large probes (cosmids, P1s, YACs) very
easy, can use a fairly standard set of conditions and map them by the
dozen on the first try.
Use a DNA probe, not RNA, we usually leave insert in the vector for small
probes and label the entire thing. Our standard is to label with biotin or
digoxygenin via nick translation, and use a fluorescent antibody sandwich
technique for maximum sensitivity in detection. In the "bad old days", we
used 3H-labelled nucs and autoradiography for detection; 32-P has
particles too energetic for microscopically accurate localization (35S has
sometimes been used, though).
Hope this was helpful, and feel free to inquire about anything which is
not clear,
Anita Hawkins
The Johns Hopkins Oncology Center
Baltimore, Maryland
