Slot blot method for the quantification of DNA sequences and mapping of chromosome rearrangements: application to chromosome 21.

As an alternative to the methods of gene dosage based on either RFLP studies or Southern blots using specific and reference probes, we designed a "slot blot" method for the evaluation of the copy number of unique chromosome 21 sequences. Varying amounts of denatured DNA from a normal control, a trisomy 21 patient, and the subject to be analyzed were loaded on the same membrane. Successive hybridizations with reference probes and chromosome 21 probes were then carried out. Intensities of the signals on autoradiograms were quantified by densitometric scanning. Graphic and statistical analysis of the linear regressions between reference and chromosome 21 probe signals were performed, and the conclusion that the DNA from the studied subject had two or three copies for a given chromosome 21 sequence was assessed by statistical comparison of the slopes. As a test for the validation of this method, 10 coded blood DNAs from five normal controls and from five trisomy 21 patients were analyzed, by using two reference (COL1A1 and COL1A2) and two chromosome 21 (D21S11 and D21S17) probes. Among the 10 DNAs analyzed, it was possible to diagnose, with 100% accuracy, normal controls and trisomic 21 individuals. Application of this methodology to the mapping of partial chromosome 21 rearrangements is presented.