In situ fluorescent nick translation procedure for plant chromosomes

Abstract

Pectinase and cellulase, which are used to macerate plant material, always show traces of DNase activities that result in DNA nicking. Moreover, the DNA polymerase I usually applied in the in situ nick translation techniques shows both 5' to 3' and 3' to 5' exonuclease activities. As a result, significant nonspecific labeling appears in control preparations that are not digested by a restriction endonuclease. Our procedure includes blocking nonspecific nick labeling before incubation with restriction enzymes (HpaII and HaeIII). This is achieved by incorporation of ddGTP into DNA by the Taq polymerase which lacks 3' to 5' exonuclease activity. This method gives satisfactory results because it eliminates nonspecific nick translation signals that are present after applying the methods described for animal material.

The mechanisms determining the nucleolar‐organizing regions inactivation of domestic horse chromosomes

“…In situ nick‐translation was performed using previously described method (Luchniak et al. 2002b) with several modifications.…”

“…In situ nick-translation was performed using previously described method (Luchniak et al 2002b) with several modifications. Metaphase spreads were dehydrated with the series of 70%, 80% and 99.5% ethanol, 5 min in each solution, and air dried.…”