DETECTION OF GENOMIC ALTERATIONS BY MOLECULAR COMBING


Year of Award:
1999
Award Type:
R21
Project Number:
CA081721
RFA Number:
PAR-98-067
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
BENSIMON, AARON
Other PI or Project Leader:
N/A
Institution:
PASTEUR INSTITUTE
In a process called 'Molecular Combing' DNA molecules attached at one end to a microscope slide are extended and aligned by a receding air-water interface and left to dry on the surface. The local action of the interface is the same on each of the molecules in solution: they are stretched in a reproducible manner to a constant value of 2kb/mum. Simple, controlled and reproducibleoptical mapping of total genomic DNA is made possible by applying Fluorescent Hybridization (FISH) or Immuno-Fluorescence (IF) to combed DNA. This technique using total human DNA easily prepared in agarose blocks allows combing of very high density of DNA molecules (103 genomes per slide) in a uniform and parallel fashion. These properties along with the scoring of single molecules allows for thorough staistical analysis of the hybridized clone sizes and distances yielding precise measurements. A physical map with a precision of a few kilobases (kb), can be obtained in this way, with no additional information from other techniques. An immediate application of screening of genomic DNA from patients for microdeliyions and translocation brekpoint at specific tumor susceptibility. The high level of resolution (2kb) within a range (5 - 300) not covered by the current methods (PCR, CGH) allows for more accurate diagnosis of specific loci. Molecular Combing is also more sensitive than CGH for the detection of low level amplifications (2-15) copies. Emphasis is now on automation in order to speed up data collection and analysis for high-throughput applications and dissemination of this of this novel technology. A second, more exploratory approach consists in scanning whole genomes of normal, precancerous cells for abnormal patterns of DNA replication units (replicons). Efficient DNA replication, dictated by regular replicon size, is necessary to maintain stable genomes. Many, if not most cancer cells show mutations in genes controlling the G1 phase of cell cycle, consequences of which might be a decrease of replication efficiency during the following S phase and increase in genomic instability. We will test this hypothesis by measuring replicon size using molecular combing in normal and transformed cells whose replication origins have been marked with bromo-deoxyuridine (BrdU). Deviation from standard values will be assessed as a general marker for genomic instability and malignancy.