New rapid multicolor PRINS protocol.
In the multiple-color primed in situ labeling (multi-PRINS) technique, using ddNTPs between two PRINS reactions can block the free 3'-end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction, using it as a primer to perform spurious elongation at nondesired sites. However, by omitting the blocking step and taking advantage of the color mixing, we developed a simple and rapid multi-PRINS technique to simultaneously detect three chromosomes in the same cell. With this protocol, one can create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-labeled dUTP) and two fluorochromes (fluorescein and rhodamine). The signals at the centromeres of three different chromosomes displayed perfect yellow, red, and green colors, respectively. The entire procedure could be completed in less than 90 min because the blocking step was omitted. This protocol is practical and efficient for multi-PRINS so that even more than three chromosome targets could be detected in the same cell.
Yan
Ju
J
Servece of Genetics, Department of Pediatrics, Faculty of Medicene and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada.
Gadji
Macoura
M
Krabchi
Kada
K
Drouin
Régen
R
D002877:Chromosomes, Human / Q000235:genetics; D004247:DNA / Q000032:analysis; D006801:Humans; D020401:Primed In Situ Labeling / Q000379:methods*; D013997:Time Factors
D004247:DNA