M. C. Roslaniec, J. C. Martin, R. J. Reynolds, L. S. Cram
Los Alamos National Laboratory, Los Alamos, NM 87545
We are developing a High Speed Optical Chromosome Sorter based on selective, irreversible photoinactivation of unwanted chromosomal DNA. Chromosomes will be analyzed as in conventional flow cytometry but no droplets will be generated. After analysis, unwanted chromosomes will be irradiated with a high power laser designed to impart photoinactivation. When desired chromosomes pass this photoinactivation point, the laser beam is interrupted by an optical modulator. The desired chromosomes are not photoinactivated and will subsequently be cloned. This method of chromosome sorting is an extension of the 'zapper' principle in which selectively photodamaged cells do not survive when placed in culture.[l] We expect optical chromosome selection rates of >1000 s-l for analysis rates of 50,000 s-l, fifty times that possible with conventional sorters.
We have successfully demonstrated photoinactivation of GM130 chromosomes in a flow cytometer. Several methods of photoinactivation are available including direct far UV damage, photosensitization via oxygen dependent and independent mechanisms or both. In our system, the DNA inactivation step is based on photoadduct formation between a psoralen derivative and chromosomal DNA. Prior to sorting, GM130 chromosomes are incubated (under dark conditions) with trimethylpsoralen. Once in the flow system, unwanted chromosome/psoralen complexes are irradiated with a UV beam from a high power argon ion laser.
Our ultimate goal is the construction of chromosome specific libraries, hence, we are using the s-Cos-l vector to examine the effects of photodarnage on cosmid cloning. Using this cosmid cloning system, the average size of packaged insert DNA is 35-45 kbp. Each irradiated chromosome/psoralen complex is estimated to receive a UV exposure of z 10 kJ/m. With psoralen and this UV exposure, we are able to form sufficient lethal photoadducts per unit insert to reduce cloning efficiencies of GM130 chromosomal DNA to <5 % while maintaining the clonability of unirradiated DNA.
Supported by the National Flow Cytometry Resource, NIH grant RR01315 and by the U. S. Department of Energy
(1) Keij, J. F.; Groenewegen, A. C.; Dubelaar, G. B. J.; Visser, J. W. M. Cytometry 1995, 19, 209-216. High-Speed Photodamage Cell Selection Using a Frequency-Doubled Argon Ion Laser.
(2) Evans, G. A.; Lewis, K.; Rothenberg, B. E. Genome 1989, 79, 9-20. High Efficiency Vectors for Cosmid Microcloning and Genomic Analysis.
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