Combined use of PRINS and FISH in the study of the dystrophin gene.
The efficacy of fluorescence in situ hybridization (FISH) may be limited in specific applications by low-resolution sensitivity. Primed in situ labeling (PRINS) is based on specific hybridization of an unlabeled oligonucleotide with a denatured template and synthesis of a single-strand DNA in situ. This method may represent a powerful alternative to FISH for gene mapping because of its ability to generate multiple independent signals within the same gene segment. We investigated the specificity of signals produced by a modified PRINS protocol combining a centromeric probe for the X-chromosome with specific primers for 3'- and 5'-terminal regions of the dystrophin gene. In approximately 70% of nuclei from male and female subjects, we detected one or two large signals (X-chromosome centromere) and two or four smaller signals (the two regions of the dystrophin gene). Specific hybridization of the oligonucleotides on Xp was demonstrated by localization of the smaller (dystrophin) and larger (X-centromere) signals on the same chromosome. Simultaneous hybridization with a centromeric probe and gene-specific oligonucleotides allowed localization of PRINS signals, and assessment of the specificity of the primers used for hybridization. This approach could facilitate identification of female carriers of small intragenic deletions in the dystrophin gene.
Cinti
Caterina
C
Istituto di Citomorfologia Normale e Patologica, CNR, Bologna, Italy. cinti@area.bo.cnr.it
Stuppia
Liborio
L
Maraldi
Nadir Mario
NM
D001483:Base Sequence; D017931:DNA Primers; D016189:Dystrophin / Q000235:genetics*; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D020401:Primed In Situ Labeling / Q000379:methods*; D014960:X Chromosome
D017931:DNA Primers; D016189:Dystrophin