Introduction

Using sequential labeling of different chromosome targets, the multiple color primed in situ labeling (multi-PRINS) technique can simultaneously and specifically display different chromosomes with different colors in the same metaphase or interphase nucleus (1-5), which can be particularly useful when a rapid screening for chromosomal abnormality is desired and the number of analyzable metaphases is limited. The first procedure developed to specifically identify different chromosome targets required a blocking treatment between two standard PRINS reactions. The incorporation of dideoxynucleotide triph-osphates (ddNTPs) acts to block the free 3'-end of the DNA strands generated during the previous PRINS reaction, thus avoiding the subsequent PRINS

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed. Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

reaction from using it as a primer to perform spurious elongation at nondesired sites (3,4,6). In practice, however, the blocking procedure does not always seem effective. In 1996, Coullin et al. (7) reported a similar technique to PRINS called HISOMA (hybridization of labeled oligonucleotides). In their technique, they used specifically labeled primers to study the a-satellite DNA of chromosome 1. This technique has been performed successfully on cytogenetic preparations from human heteroploid and human x hamster hybrid cell lines in addition to frozen tissue sections. The use of different fluorochromes and the possible combination with an unlabeled elongation in 3' of the oligonucleotides, which stabilize its hybridization, led to a simple multicolor method (7).

In the double-PRINS technique, to specifically detect two different chromosomes without the blocking step, we found that the correct identification easily can be performed because the signals obtained from the last PRINS reaction always show a single original color. From this finding, we developed a multi-PRINS technique (8) by omitting the blocking step and taking advantage of mixing two fluorochromes (fluorescein and rhodamine) to create a third color for the detection of three different chromosome targets (Figs. 1 and 2). By selecting the labeling order, either in bio-digoxigenin (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/avi-din-rhodamine, the signals at the centromeres of three different chromosomes displayed perfect yellow, red, and green colors, respectively. Because the blocking step is omitted, the entire procedure can be completed in less than 90 min. We showed that this is a practical and efficient way to conduct multi-PRINS so that even more than three chromosome targets could be detected in the same cell.

This technique has been used successfully to determine the copy numbers of different chromosome targets from interphase nuclei in peripheral blood and bone marrow (9,10) and in amniotic fluid cells (Gadji et al., unpublished data). We also have recently developed a double-strand PRINS technique (11). A high labeling efficiency of human telomeres was obtained by using two primers, (TTAGGG)7 and (CCCTAA)7, to label both forward and reverse telomeric DNA strands (Fig. 3). On the basis of the principle of this multi-PRINS technique, four sequential multi-PRINS reactions were performed successfully on spermatozoa, lymphocytes (12), oocytes, and polar bodies (13). The following fluorochromes fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and Cascade Blue incorporated in sequential PRINS reactions allowed the rapid and distinct labeling of four chromosomes displaying distinct color spots (green, red, yellow, and blue).

Fig. 1. The schematic triple-color PRINS technique. After three sequential PRINS reactions, the target sites of chromosomes 7, 8, and 18 were identified with yellow, red, and green colors, respectively. Horizontal dashed arrows under the chromosomal DNA indicate each strand extension site from the beginning to the end of each PRINS reaction. The procedure illustrated corresponds to the bio-dig-bio labeling order using the detection mix avidin-fluorescein/anti-dig-rhodamine. A similar schema can be used to illustrate the reverse labeling order, dig-bio-dig, using the detection mix anti-dig-fluorescein/avidin-rhodamine (8).

Fig. 1. The schematic triple-color PRINS technique. After three sequential PRINS reactions, the target sites of chromosomes 7, 8, and 18 were identified with yellow, red, and green colors, respectively. Horizontal dashed arrows under the chromosomal DNA indicate each strand extension site from the beginning to the end of each PRINS reaction. The procedure illustrated corresponds to the bio-dig-bio labeling order using the detection mix avidin-fluorescein/anti-dig-rhodamine. A similar schema can be used to illustrate the reverse labeling order, dig-bio-dig, using the detection mix anti-dig-fluorescein/avidin-rhodamine (8).

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