Introduction

Primed in situ labeling (PRINS) was first applied to examine the molecular structure of human and animal chromosomes where it proved to be a useful alternative to established fluorescence in situ hybridization protocols (1,2). Initial efforts to apply this technique to plants were stimulated by a need for a simple procedure to fluorescently label specific DNA repeats on mitotic chromosomes in liquid suspension before their analysis using flow cytometry (3,4). Subsequent studies aimed at improving the sensitivity and specificity of the procedure (5), including a modification of PRINS called cycling PRINS (6,7). Despite these significant advances, the sensitivity of PRINS on plant chromosome preparations remains lower when compared with fluorescence in situ

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.

Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

hybridization, and the method does not allow for the reliable visualization of short single- and low-copy targets (8).

The most productive applications of PRINS have been the investigation of the genomic distribution of plant satellite repeats (9-11) and the analysis of flow-sorted chromosomes (12). Because of its speed, PRINS has been the method of choice for identifying flow-sorted chromosomes and for determining the purity of sorted chromosome fractions in a number of species, including garden pea (13), barley (14), and wheat (15,16). In addition, PRINS was found useful in the development of molecular karyotypes, in which labeled satellite repeats can be used as cytogenetic landmarks and allow the identification of morphologically similar chromosomes (17). Recently, it has been demonstrated that PRINS can discriminate and, thus, specifically label, closely related repeat subfamilies (11). This labeling was achieved by designing oligonucleotide primers for the reaction such that their 3'-ends corresponded to sequences specific for the individual subfamilies. Because the sequence similarity between the primer 3'-end and the target is crucial for its extension by Taq polymerase, any mismatch in this region greatly reduces its performance.

In this chapter, we provide a protocol for PRINS labeling of satellite repeats on plant chromosomes. As an example, we demonstrate labeling of VicTR-B repeats (10) on chromosomes of a legume plant Vicia grandiflora. This species genome contains several subfamilies of the VicTR-B sequences differing in short (2-4 bp) variable regions (J. Macas et al., 2005, unpublished observations). Targeting the primer to one such region allows the visualization of the chromosome distribution of that specific subfamily, whereas using a probe derived from conserved sequence domains labels all loci containing the VicTR-B repeats. The protocol involves denaturation of chromosomal DNA directly in the PRINS reaction mixture, annealing of unlabeled oligonucleotide primers to corresponding chromosomal targets, and their extension by Taq polymerase in the presence of fluorescently labeled dNTPs. Only a single cycle of DNA synthesis is required to produce sufficient signals, which makes the labeling procedure very short (approx 1 h).

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