Creating a new color by omission of 3 ' end blocking step for simultaneousdetection of different chromosomes in multi-PRINS technique

In the multiple color primed in situ labeling (multi-PRINS) technique, the blocking step using ddNTPs, incorporated by a DNA polymerase, is an importa nt procedure that blocks the free 3' end generated in the previous PRINS re action, thus avoiding the next PRINS reaction using it as a primer to perfo rm spurious elongation at non-desired sites. However, we found that omissio n of the blocking step never affected the correct identification of two chr omosomes because the signals from the second PRINS reaction site always sho wed the pure original color. Nevertheless, taking advantage of the color mi xing, we successfully used a multi-PRINS technique to create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-la beled dUTP) and two fluorochromes (fluorescein and rhodamine) in order simu ltaneously to detect three chromosomes in the same cell. By arranging the l abeling either in bio-dig-bio or in dig-bio-dig order in the sequential PRI NS reaction, then detecting with a mixture of avidinfluorescein/anti-dig-rh odamine or a mixture of anti-digfluorescein/avidin-rhodamine, the signals a t 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. We showed that this is a practical and efficient way to carry out multi-PRINS so that even more than three chromosome targets could be detected in the same cell.