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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

A High Speed Method for Chromosome Sorting Using the Photoinduced Cross-linking of Psoralen Derivatives with Chromosomal DNA: Preliminary Studies

M. C. Roslaniec, J. C. Martin, R. J. Reynolds, L. S. Cram
Life Sciences Division - 1; Los Alamos National Laboratory; Los Alamos, New Mexico, 87545

A High Speed Optical Chromosome Sorter based on selective, irreversible photoinactivation of chromosomal DNA is being developed. Chromosomes are analyzed as in traditional FCM, however, rather than relying on droplet formation, (a rate limiting factor in droplet sorting), sorting is achieved by photoinduced cross-linking of undesired chromosomes with a photosensitive compound such as psoralen. Cross-linked DNA cannot be denatured and is rendered unclonable. Desired chromosomes are not irradiated and will remain clonable providing a means of separation. Initial results indicate that treating pBluescript SK(+) phagemid DNA (100 ng) with ~ 4.0 µg of 8-methoxypsoralen and 35 kJ/m(2) UV lamp irradiation produces ~ 5 photoadditions (mono- and diadducts)/kbp. We have determined that this level of cross-linking effectively inhibits transfection of XL1-blue E. coli cells. Furthermore, since our ultimate goal is the construction of chromosome specific libraries, we are using the s-Cos- 1 vector to examine the effects of photo-cross-linking on cosmid cloning. Finally, we report preliminary results of the effect of chromosome photoinactivation in flow.

Supported by the National Flow Cytometry Resource, NIH grant RR01315 and by the U. S. Department of Energy