Application of the RB-FPG technique for distinguishing early and late replicating X chromosomes in farm animals

Abstract

The sex chromosomes of Murrah buffalo and Sahiwal cattle (females) were studied for R-banding patterns. A short term whole blood culture method was used for obtaining metaphase chromosomes. The R-banding method using 5-bromo-2'-deoxyuridine (BrdU), the fluorescent 33258 Hoechst stain followed by blue-black light exposure and Giemsa staining (RB-FPG) was applied to the chromosome preparations. The two X chromosomes in all the females were clearly distinguished. One of the chromosomes (early replicating) had alternative light and dark bands and the other X chromosome (late replicating) was either completely pale banded or had one or a few dark bands. The reason for this phenomenon could be BrdU incorporation in the late replicating X chromosome, which on exposure to blue light in the presence of 33258 Hoechst is destroyed and does not take the stain. Thus, one of the two X chromosomes shows differential staining behaviour.