Quantifying single gene copy number by measuring fluorescent probe lengthson combed genomic DNA

An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified mole cules of genomic DNA on a glass coverslip. It has the advantage that a larg e number of genomes can be combed per coverslip, which allows for a statist ically adequate number of measurements to be made on the combed DNA. Conseq uently, a high-resolution approach to mapping and quantifying genomic alter ations is possible. The approach consists of applying fluorescence hybridiz ation to the combed DNA by using probes to identify the amplified region. M easurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested f or low copy number amplifications by determining the copy number of chromos ome 21 in a normal and trisomy 21 cell line. It then was tested for high co py number amplifications by quantifying the copy number of an oncogene ampl ified in the tumor cell line GTL-16, These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sens itivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.