MICRODROPS AND FLOW SORTING FOR CHROMOSOME ISOLATION

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Flow sorting is a powerful technology for the isolation of chromosomes. Pure chromosome fractions have been essential to the human genome project. Purification of chromosome sub-fragments and some chromosomes (especially mouse) is not possible, however, due to limitations in the current technology. This project provides a means for addressing these limitations by developing methods for sequence-specific fluorescent labelling of chromosomes with DNA probes followed by flow sorting of specifically labelled chromosomes. This proposal represents the first step in the development of a simple method for the physical isolation of specific large chromosomal fragments (greater than or equal to 1 megabase) in amounts suitable for experimentation. Individual chromosomes will be encapsulated into gel microdrops (GMDs). The GMD-chromosome unit is a physically manipulable testing entity. In situ hybridization using DNA probes specific to the desired chromosome will be performed within the GMD-chromosome unit. Following specific tagging, the GMD-encapsulated chromosomes will be isolated by flow sorting. This approach combines the specificity of in situ hybridization with the power of automated separation by flow sorting. The specific goals of the research are: (i) To develop procedures for in situ hybridization within GMDs, (ii) To develop procedures for encapsulation of individual chromosomes in GMDs, (iii) To demonstrate specific labelling of chromosomes within GMDs, and (iv) To isolate specifically labeled GMD-chromosome units by flow sorting.