Use of Alu-PCR to characterize hybrids containing multiple fragments and to generate new Xp21.3–p22.2 markers

Abstract Irradiation fragment hybrids potentially provide highly enriched sources of region-specific human DNA. However, such hybrids often contain multiple human pieces, not all of which can be easily detected. To develop specific resources for rapidly generating markers from Xp21.3–p22.2, we have single cell cloned two previously constructed irradiation hybrids that contain markers in this region and have achieved segregation of the different known fragments originally retained. Alu -PCR products were generated from subclones positive or negative for Xp21.3–p22.2 markers, and comparison of the ethidium bromide patterns between sister subclones facilitated identification of bands likely to map to particular regions; in contrast, subclones that shared markers but were derived from independent lines showed no overlap in ethidium bromide pattern. All Alu -PCR products from one subclone, 50K-19E, in which only three closely linked markers were detected (DXS41, DXS208, DXS274) were mapped back to their region of origin. Of 28 products, 15 mapped to Xp21.2–p22.2, and these make up a new set of regionally assigned markers. However, the mapping data identified four separate Xp fragments in 50K-19E, only one of which had been picked up by marker analysis. Mapping back gel-isolated Alu -PCR products from an irradiation hybrid prior to any cloning or screening generates a comprehensive profile of the human DNA retained and permits rapid selection of sequences derived only from the region of interest.