Individual pairs of human chromosomes can be reliably identified by a new method that does not require special optical equipment and that results in permanent preparations. This method, which is based on treatment of the chromosomes in situ with NaOH, followed by incubation in sodium chloride-trisodium citrate and Giemsa staining, results in highly specific banding patterns in characteristic regions of the chromosome arms. It should prove useful for the detection of small structural changes in chromosomes.During the past 15 years remarkable progress has been made in the descriptive morphology of both normal and abnormal human chromosomes, but with conventional cytological and autoradiographic techniques we have thus far failed to identify every chromosome in the human complement. Furthermore, these techniques have revealed very little differentiation within a chromosome arm, so that structural rearrangements that do not alter the length or centromere position, or produce only slight changes in these, will escape detection.Since the original description of specific fluorescent staining of human chromosomes (1), investigations have led to renewed hope for subdividing the classical chromosome groups into individual pairs. This paper describes a new method of differentiating human chromosomes that does not require fluorescence microscopy and that results in permanent preparations. The chromosomes exhibit banding patterns in specific regions. In addition to the sex chromosomes, 20 of the 22 autosomal pairs have been identified by this technique, and the remaining two are tentatively classified. MATERIALS AND METHODSLymphocyte and fibroblast cultures from normal persons were prepared and harvested in the usual manner. Colchicine, 0.04 /Ag/ml of medium, was added to the cultures 2 and 6 hr before harvest. After hypotonic treatment with 1% sodium citrate and fixation in methanol-acetic acid 3:1, flame-dried slides were prepared. The method to be described consists of treatment of the chromosomes in situ with NaOH, followed by incubation in several concentrations of a saline-citrate solution (SSC). Specific regions of the chromosome arms then stain differentially with buffered Giemsa. Because a number of parameters have varied in our pilot experiments and we do not yet know the optimal conditions for treatment, we will present first a basic procedure that we have found to produce bands fairly consistently, and then a range of times and concentrations that have been tested.The slides are treated for 30 sec in a solution of 0.07 N NaOH in 0.112 M NaCl (pH 12.0) at room temperature and then rinsed three times in 12 X SSC (pH 7.0) for 5-10 min each time. They are then incubated in 12X SSC at 650C for 60-72 hr. After incubation, the slides are passed through three changes of 70% ethanol and three changes of 95% ethanol (3 min each). After air-drying, slides are stained for 5 min in buffered Giemsa solution (pH 6.6), rinsed briefly in distilled water, air-dried again, and mounted in Permount.The alkaline solution is pr...

Variation in the length of the Y chromosome in a population of 100 males, derived from five different racial and ethnic sub-groups, was investigated. The Y chromosome and selected autosomes were measured in 1000 metaphase cells. Statistical analysis of the data demonstrates significant differences in mean length of the Y among some of the groups as well as among the individuals within each group. The mean length of the Japanese Y chromosome was significantly greater than that of all other groups. Among a sample of 736 circular metaphase plates, the Y chromosome was peripherally located in 241 cells.

Genetic polymorphism has been demonstrated in man for many characteristics including blood groups, serum proteins, tissue enzymes, and hemoglobins. A class of chromosomal polymorphism involving constitutive heterochromatin has now been found. Through the use of a special technique that permits visualization of heterochromatin, seven heterochromatin variants have been found among four individuals. These results suggest a very high frequency of variability of heterochromatin in the population.

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