Direct visual resolution of gene copy number in the human photopigment gene array

PURPOSE. To visualize by direct fluorescent in situ hybridization the entir e human visual pigment gene array on single X-chromosome fibers and to comp are the results with values obtained by other molecular techniques. METHODS. The size of the opsin gene array on the X-chromosome in eight male subjects was investigated by (i) direct visual in situ hybridization (DIRV ISH) on elongated DNA fibers; (ii) quantitation of genomic restriction frag ments after Southern blot hybridization; (iii) quantitation of restriction fragment length polymorphism after PCR amplification (PCR/RFLP); and (iv) s izing of NotI fragments by pulsed field gel electrophoresis and Southern bl ot detection. Each male subject's color vision was assessed by Rayleigh mat ches on a Nagel Type 1 anomaloscope. RESULTS. The number of genes resolved by the DIRVISH protocol, which ranges from 1 to 6, agrees exactly with the gene array sizes obtained in the same male subjects from pulsed field gel electrophoresis, but differs from the estimates derived from the commonly used indirect Southern blot hybridizati on and PCR/RFLP quantitation methods. In particular, the PCR/RFLP method ov erestimates the copy number in all but the smallest arrays. CONCLUSIONS. Visualization of the X-chromosome opsin gene array by DIRVISH provides a new, direct method for obtaining exact copy numbers and helps to resolve the controversy about the range and the average visual pigment gen e number in the human population in favor of smaller average array sizes.