In situ fluorescent nick translation procedure for plant chromosomes.
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.
Luchniak
P
P
Department of Cytogenetics and Plant Molecular Biology, University of Lodz, Poland.
Maluszynska
J
J
Olszewska
M J
MJ
D002875:Chromosomes / Q000648:ultrastructure; D004396:Coloring Agents; D027468:Crepis / Q000648:ultrastructure*; D004256:DNA Polymerase I / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000737:chemistry; D018744:DNA, Plant / Q000737:chemistry / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D006651:Histocytochemistry; D020287:In Situ Nick-End Labeling / Q000379:methods*; D008856:Microscopy, Fluorescence; D010944:Plants / Q000648:ultrastructure*
D004396:Coloring Agents; D018744:DNA, Plant; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes