Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

Abstract

Summary. The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.

Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes

“…In double hybridization expetiments performed under a variety of less stringent hybridization conditions (not shown) we found, however, that lc was still largely confined to lq12, whereas 18c hybridized to the centromeric regions of most chromosomes [3]. The more extensive cross-hybridization of 18~ probe is likely to account for the fact that in both normal diploid and glioma cell cultures nuclei with spot numbers above the peak fraction were generally somewhat more numerous for 18~ as compared to lc demonstrates six chromosomes decorated with the biotinylated 18~ probe.…”

“…merit regions of other chromosomes such as chromosome 9 in case of lc and chromosome 2 in case of 18c indicated some cross-hybridization to related sequences in the constitutive heterochromatin of these other chromosomes [3,27,311. Generally these minor signals were easily distinguished on the basis of intensity.…”

“…It should be noted that the stringency of hybridization and posthybridization washes is crucial for detection of "chromosome specific" labeling by lc and 18c DNA probes. These DNA inserts will hybridize to several chromosomes in addition to chromosomes 1 and 18 with reduced formamide concentrations, e.g., at 40% formamide [3,27,311. Hybridization mixtures with formamide concentrations higher than 60%, or posthybridization washes at higher temperatures, yielded weaker but not significantly more specific signals.…”

“…The detection of chromosome abnormalities in interphase nuclei, an approach termed "interphase cytogenetics" [3], appears to be a useful addition to the classical metaphase analysis. The examination of numerical aberrations in interphase nuclei can yield a first approximation of specific chromosomal aberrations in large populations of tumor cells.…”

“…Discussion). Repeated DNAs often define large chromosomal domains and subsets of the centromeric alphoid repeat family [21- [3,211). Despite this limitation it has been possible to diagnose trisomy 21 [34] and trisomy 18 [3] in interphase nuclei of amniotic fluid cells.…”