Chromosome abnormalities produce a variety of clinical phenotypes including malformation and mental retardation. This is a significant factor in human mortality and morbidity because some 0.7% of live-born children have congenital malformations owing to chromosome abnormalities. Fine study of the chromosomes is thus crucial in the analysis of most congenital diseases, with implications for clinical management and counseling.

For nearly 20 years, fluorescence in situ hybridization (FISH) has been an important and useful adjunct to the cytogenetic evaluation of chromosome rearrangements and gene deletions. FISH allows resolution of DNA sequences separated by only a few megabases in metaphase or prophase chromosomes; the low-end

From: Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina © Humana Press Inc., Totowa, NJ

resolution is approximately 20 kb. However, production of locus-specific FISH probes is complicated, expensive, and time consuming, and many of the probes are not readily available.

We are concerned here with a powerful new method—primed in situ labeling (PRINS)—that can be used as an alternative to FISH (1,2). With PRINS, simple oligonucleotide primers are employed instead of cloned DNA probes for the in situ labeling of chromosomal loci. According to the PRINS method, unlabeled oligonucleotide primers are annealed in situ to complementary target sequences on chromosomes and extended in the presence of labeled nucleotides and DNA polymerase. Chromosomal DNA acts as template for the extension and the labeled nucleotides as substrate for the DNA polymerase. The labeled extended target is visualized by fluorescence microscopy.

Although most early applications of the method dealt with repetitive sequences, PRINS could also be used to identify single-copy genes. In fact, Cinti et al. (3) used PRINS to identify the X-linked factor IX gene more than 10 years ago. That was the first published example of identification of a single-copy gene by PRINS, and it represents a significant biologic milestone, because every known gene sequence is a potential primer source. It follows that any gene or exon could be located in chromosome preparations in situ, provided that a unique nucleotide sequence is available. Further development and adaptation of the PRINS method could have a fundamental impact on medical genetics. The physical mapping of individual loci would now be possible in most laboratories concerned with the study of human chromosomes and the diagnosis of genetic diseases.

Here we describe the PRINS method with special emphasis on the detection of unique sequences and single-copy genes such as SRY and SOX3 (4).

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