John M. S. Bartlett.pdf - Bio-Nica.info

PRINS and Immunocytochemistry 453
63
Primed In Situ Nucleic Acid Labeling Combined with
Immunocytochemistry to Simultaneously Localize DNA
and Proteins in Cells and Chromosomes
Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman
1. Introduction
In the past decade, the primed in situ (PRINS) labeling technique has become an
alternative to fluorescence in situ hybridization (ISH) for the localization of nucleic acid
sequences in chromosome, cell, and tissue preparations (1–8). The PRINS method is
based on the rapid annealing of unlabeled primers (restriction fragment, PCR product,
or oligonucleotide) to complementary target sequences in situ. These primers serve
as initiation sites for in situ chain elongation using Taq DNA polymerase and labeled
nucleotides. Incorporated fluorochrome-labeled nucleotides can be detected directly by
fluorescence microscopy, whereas haptenized (e.g., biotin, digoxigenin, dinitrophenyl)
nucleotides can be visualized by the additional application of fluorochrome- or
enzyme-conjugated avidin or antibody molecules (4,5,9,10), followed by fluorescence
microscopy or brightfield visualization of enzyme reaction products. Particularly,
rapidity, simplicity, and cost-effectiveness have made the PRINS technique a useful
tool in cytogenetics and cell biology. Its detection sensitivity, however, seems to
be limited to repetitive targets for a long time. Only recently, the combined use of
multiple oligonucleotides for 1 locus together with tyramide signal amplification
have shown the first reproducible results demonstrating single-copy gene detection
by PRINS (11).
With immunocytochemistry (ICC), specific information can be obtained regarding
the presence or absence of proteins or antigens in chromosomes, cells, and tissue
sections, thus allowing one to characterize the function of structural proteins in
chromosomes or to phenotype cells (for example, their type of differentiation and
proliferative activity). In addition, protocols have been developed to efficiently combine
protein and nucleic acid detection in the same biological material to, for example,
immunophenotype cells harboring a specific chromosomal aberration or viral infection,
determine cytokinetic parameters of tumor cell populations that are genetically of
phenotypically aberrant, and study the structural organization of chromosomes and the
cell nucleus (10,12). The success and sensitivity of such a combined procedure depends
on factors, such as preservation of cell morphology and protein epitopes, accessibility
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
453