Introduction

For nearly two decades, fluorescence in situ hybridization (FISH) has been a useful and significant adjunct to the cytogenetic evaluation of gene deletions and subtle chromosome rearrangements. However, in recent years, a new method, primed in situ labeling (PRINS) has been applied to the localization of a-satellite sequences and to the gross identification of individual chromosomes (1,2). According to the PRINS method, laboratory-synthesized oligonucleotide probes (i.e., PRINS primers) are used instead of cloned DNA for the in situ localization of individual genes. The PRINS primers are annealed to complementary target sequences on chromosomes and are extended in the presence of labeled nucleotides (3) utilizing Taq DNA polymerase. Chromosomal DNA acts as template for the extension and the labeled nucleotides as substrate for the DNA polymerase.

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

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

Although PRINS initially was used to detect repetitive sequences, such as the a-satellite, Alu, and telomeric repeats (1,2,4), the method also could be used to identify a single copy sequence, the X-linked factor IXgene (5). That work represents a significant biological milestone because every known gene sequence is a potential primer source and, thus, potentially is amenable to fine analysis by the PRINS method.

We modified the PRINS method for increased sensitivity and applied the TSATM Biotin System to localize the sex-determining SRYgene in XX men, in a woman with XY gonadal dysgenesis, and in an azoospermic man with Xp-Yp interchange. Modifications included the use of day-old slides, use of 0.02 N HCl to remove loosely bound protein, the application of supernumerary primers for a single target locus, single-step annealing and extension, the use of TaqStart monoclonal antibody, and stringent washing in standard saline citrate (SSC) to minimize background. Our results enabled visual confirmation of the molecular data and precise localization of the SRY gene in all cases (6). In other studies (7), we mapped the SOX3 gene by using the same methods. It follows that further development of the PRINS method may allow localization of any gene or exon in chromosome preparations in situ, provided that the nucleotide sequence is available.

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