NDLI: Creating a new color by omission of 3′ end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique

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Creating a new color by omission of 3′ end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique

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Creating a new color by omission of 3′ end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique

Content Provider	Springer Nature Link
Author	Yan, Ju Bronsard, Marc Drouin, Regén
Copyright Year	2000
Abstract	In the multiple color primed in situ labeling (multi-PRINS) technique, the blocking step using ddNTPs, incorporated by a DNA polymerase, is an important procedure that blocks 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 non-desired sites. However, we found that omission of the blocking step never affected the correct identification of two chromosomes because the signals from the second PRINS reaction site always showed the pure original color. Nevertheless, taking advantage of the color mixing, we successfully used a multi-PRINS technique to create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-labeled dUTP) and two fluorochromes (fluorescein and rhodamine) in order simultaneously to detect three chromosomes in the same cell. By arranging the labeling either in bio-dig-bio or in dig-bio-dig order in the sequential PRINS reaction, then detecting with a mixture of avidin-fluorescein/anti-dig-rhodamine or a mixture of anti-dig-fluorescein/avidin-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. 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.
Starting Page	565
Ending Page	570
Page Count	6
File Format	PDF
ISSN	00095915
Journal	Chromosoma
Volume Number	109
Issue Number	8
e-ISSN	14320886
Language	English
Publisher	Springer-Verlag
Publisher Date	2000-12-20
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Content Type	Text
Resource Type	Article
Subject	Genetics Genetics (clinical)

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Creating a new color by omission of 3′ end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique

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