Laser-assisted microdissection and sorting of reversibly immobilized metaphase chromosomes

Chromosome microdissection is a very valuable approach for the construction of detailed specific chromosome libraries. Metaphase chromosomes from both murine and human origin were immobilized by centrifugation onto a glass coverslip previously coated with a thin matrix. Reversible immobilization was obtained using an alginate matrix which exhibits a sol-gel transition in the presence of a counter-ion and a chelating agent. The matrix was selected for its optical qualities permitting imaging, chromosome banding and laser-assisted micromanipulations using focused laser beams. Whole chromosomes and chromosome fragments were cut and released from the matrix using the LaserScissors, a laser microdissecting system mounted onto an inverted microscope. Chromosomes or chromosome fragments were then transported towards the tip of a glass micropipette by means of optical trapping using the LaserTweezers. They were recovered individually with the CellSelector, a microsyringe pump, and made available for DNA amplification using PCR. We are currently in the process of characterizing specific chromosome fragments. Routine recovery of single chromosomes and sub-chromosomal regions should find applications for the mapping of genomes of several species including human and mouse. Reversible immobilization procedures and laser assisted micromanipulations of metaphase chromsomes should be beneficial to the Human Genome Project.