Detection of in situ hybridization to human chromosomes with the atomic force microscope.
Atomic force microscopy (AFM) permits one to generate a topographic representation of the sample under investigation with high spatial resolution. We assumed that cytochemical staining techniques, which yield reaction products which can be discriminated from the surrounding material on basis of their topographic properties, would be applicable in AFM. Here we show the validity of this assumption by employing an in situ hybridization technique in which the final label was the precipitated product of a peroxidase/diaminebenzidine reaction. After hybridization of the DNA probe pUC1.77 that recognizes the heterochromatic region of human chromosome 1 (1q12), the AFM clearly detects the sites of in situ hybridization. In situ hybridization with DNA probe p1-79 results in clear marking of the telomere region 1p36. The diameter of the probe p1-79 linked reaction product was 75-100 nm, indicating that resolution of 200 nm can readily be reached with this AFM approach of DNA mapping. This precision is directly linked with the amount of precipitated material.
Putman
C A
CA
Department of Applied Physics, University of Twente, Enschede, The Netherlands.
De Grooth
B G
BG
Wiegant
J
J
Raap
A K
AK
Van der Werf
K O
KO
Van Hulst
N F
NF
Greve
J
J
D002877:Chromosomes, Human / Q000648:ultrastructure*; D015342:DNA Probes; D006801:Humans; D017403:In Situ Hybridization; D008853:Microscopy / Q000295:instrumentation / Q000379:methods*
D015342:DNA Probes