Both the primed in situ (PRINS) labeling and the peptide nucleic acid (PNA) techniques constitute alternatives to the fluorescence in situ hybridization (FISH) for chromosomal screening and aneuploidy detection.

The PRINS labeling is based on the use of short specific primers for repeated DNA sequences. An advantage of primers is their ability to differentiate between closely related sequences (1,2). This feature has been used for generating chromosome-specific primers from the centromeric a-satellite DNA motifs (3,4). Since its introduction, the PRINS procedure has been considerably improved and simplified, with the development of sequential multicolor protocols and the direct use of fluorochromes. Numerous applications of PRINS have been described in humans, mammals, fish, insects, and plants, demonstrating that the PRINS procedure can be easily adapted to various types of cells (5-7).

The PNAs are a new class of probes recently introduced in molecular cyto-genetics. These molecules are synthetic nucleic acids analogs in which the phosphodiester backbone is replaced by a noncharged peptide-like backbone (8). This unique structure gives PNAs the capacity to hybridize to complemen-

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

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

tary RNA and DNA sequences with high affinity and specificity and a great resistance to nucleases and proteinases. The remarkable physicochemical properties of PNAs have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy, genome mapping, and mutation detection (9). During the last few years, the use of PNAs has proven its powerful usefulness in cytogenetics. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, and spermatozoa, as well as on isolated oocytes and blas-tomeres, indicating that PNAs could become a valuable tool for in situ chromosomal investigations (10-12).

PRINS and PNA present several features that make them very attractive for cytogenetic purposes. These two techniques can advantageously be combined on a same cell preparation. This combined use opens up interesting possibilities for multiplex assays. This chapter describes this innovative procedure for in situ detection of several chromosomes.

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