Laser-microdissection of chromosome fragments: Use for making reagents for the diagnosis of cytogenetic anomalies and caryotypes improvement
Inventor: PHILIPPE METEZEAU
Description of invention:
The analytical and preparatory cytometry section of the Institut Pasteur finalizes a precise, rapid and reproducible method to obtain chromosome fragments of variable size (from 1.5 Megabase to about 30 Megabase).
This methodology avoids the disadvantages of conventional methods : flow cytometry, unable to distinguish between two chromosomes with the same amount of DNA and the same AT/GC ratio, and microdissection by micro-pippeting, requiring a "scratching" stage which might damage the DNA quality. Moreover, it also has advantages relating to the quality of probes obtained, the execution speed and the possibility to keep the original matrix to avoid the loss of a precious material and allow reproduction. Moreover, the specific conditions of PCR allow DNA amplification with only one copy, thus it ensures the homogeneity of the chosen probe.
This technology requires a laser beam to destroy by burning, on metaphase plates, all of non desired chromosome or chromosome fragments.
The fragment which has not burn is isolated and can be easly recovered.
Its DNA, amplified by PCR and cloned in a plasmid, allows either the production of specific probes, usable for fluorescent in-situ hybridization (FISH), or analysis of suspected fragments in the context of a medical diagnosis search for a gene.
With such a method, several types of probes can be obtained, either known chromosomic probes, or chromosomic DNA from an unknown origin which we want to identify.
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