Article

Analysis of DNA replication parameters in isolated organs of drosophila melanogaster by molecular combing

Cell and Tissue Biology 02/2011; 5(1):1-8. DOI: 10.1134/S1990519X11010081

ABSTRACT Methods of DNA physical mapping and the direct visualization of the replication and transcription in specific genome regions
play a crucial role in the study of structural and functional organization of eukaryotic genomes. Since DNA strands in cells
are organized into high-fold structure and structurally present as highly compacted chromosomes, the majority of methods at
the chromosome level are of low-resolution. An approach to increasing the resolution and accuracy is molecular combing. The
method is based on the process of stretching and alignment of DNA molecules that are covalently attached with one of the ends
to the coverglass surface. In this article, we describe the major methodological steps of molecular combing and their adaptation
to study the parameters of DNA replication in polyploid and diploid tissues of drosophila larvae.

Keywordsmolecular combing–replication–polyploid tissues–drosophila

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    ABSTRACT: Molecular combing technology is an important new tool for the functional and physical mapping of genome segments. It is designed to identify amplifications, microdeletions, and rearrangements in a DNA sequence and to study the process of DNA replication. This technique has recently been used to identify and analyze the dynamics of replication in amplified domains. In Bradysia hygida, multiple amplification initiation sites are predicted to exist upstream of the BhC4-1 gene. However, it has been impossible to identify them using the available standard techniques. The aim of this study was to optimize molecular combing technology to obtain DNA fibers from the polytene nuclei of the salivary glands of B. hygida to study the dynamics of DNA replication in this organism. Our results suggest that combing this DNA without prior purification of the polytene nuclei is possible. The density, integrity, and linearity of the DNA fibers were analyzed, fibers 50 to 300 kb in length were detected, and a 9-kb fragment within the amplified region was visualized using biotin detected by Alexa Fluor 488-conjugated streptavidin technique. The feasibility of physically mapping these fibers demonstrated in this study suggests that molecular combing may be used to identify the replication origin of the BhC4-1 amplicon.
    Genetics and molecular research: GMR 01/2012; 11(3):2060-70. · 0.99 Impact Factor

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