Use of Alu-PCR to characterize hybrids containing multiple fragments and to generate new Xp21.3-p22.2 markers.
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.
Benham
F
F
Department of Genetics and Biometry, University College London, United Kingdom.
Rowe
P
P
D001483:Base Sequence; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D005819:Genetic Markers; D006801:Humans; D006822:Hybrid Cells / Q000528:radiation effects / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014960:X Chromosome / Q000528:radiation effects
D015342:DNA Probes; D005819:Genetic Markers; D004247:DNA