PP23. THE BASIC AND PRACTICAL IMPLICATIONS OF QUANTITATIVE FLUORESCENCE IN SITU HYBRIDIZATION (QFISH)
IOUROV I.Y.1,2, Vorsanova S.G.1,2, Monakhov V.V.1,2, Soloviev I.V.1, Kurinnaya O.S.2, Yurov Y.B.1,2 1. National Research Center of Mental Health, RAMS, Moscow, Russia; 2. Institute of Pediatrics and Children Surgery, Roszdrav, Moscow, Russia. e-mail: y_yurov@yahoo.com
*Corresponding Author:
page: 58

Abstract

A recently developed approach to quantify fluorescence in situ hybridization (FISH) signals (Iourov et al., 2005) possesses the potential to improve the resolution of molecular cytogenetic studies by FISH-based techniques. We were able to show that analysis of chromosome parental origin by QFISH is as efficient as corresponding molecular genetic techniques in studying non-disjunction of chromosome 21 (Vorsanova et al.,2005) and X-chromosome inactivation. Next, QFISH was applied to differentiate between centromeric associations and chromosome loss in interphase FISH studies with centromeric DNA probes (essential technique for investigation of chromosomal mosaicism) (Soloviev et al., 1997; 1998; Yurov et al., 1996; 2002). We found that among 2-9% of nuclei of cultured lymphocytes and chorionic villi cells demonstrating one FISH signal over 80% were those that are characterized by centromeric associations. It strongly indicates that the rate of chromosome loss obtained without QFISH might be artificially 5-10-times increased as to real one being not over 0.2-1.2% per DNA probe. Furthermore, the kinetic analysis of FISH reactions by QFISH offered the possibility to address the size of highly repetitive DNA blocks of chromosomes 1, 9, and 10 using probes of known genomic DNA target and size as a marker. Thus, the size of 1qh was found to vary from 12.7 to 29.4 Mb in different individuals, 9qh from 6.5-11.2 Mb, and in case of 10cen (alpha satellite DNA) it was about 2.2 Mb in all the cases. It should be noted that heterochromatic regions of chromosomes 1 and 9 have lacked to date the DNA size definitions. Together, our data demonstrate that quantitative FISH (QFISH) represents an efficient additional tool for molecular cytogenetics. The work was supported by INTAS 03-51-4060.




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