Molecular combing in the analysis of developmentally regulated amplified segments of Bradysia hygida

Abstract

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 2061 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 11 (3): 2060-2070(2012 Molecular combing analysis in Bradysia hygida 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.

Chromosomal localization and partial sequencing of the 18S and 28S ribosomal genes from Bradysia hygida (Diptera: Sciaridae)

“…Bradysia impatiens has been reported to be an economically problematic species in Florida (Mead, 1978), Bradysia species are known major insect pests in greenhouses (Meers and Cloyd, 2005), and Bradysia odoriphaga Yang and Zhang is the most serious pest of Chinese chive (Li et al, 2007). Bradysia hygida, first described by Sauaia and Alves (1968), is a sciarid that has been extensively used as a model system to study developmentally regulated gene transcription and amplification (Laicine et al 1984;Monesi et al 1995;Fiorini et al, 2001;Basso Jr et al, 2002;Candido-Silva and Monesi, 2010;Garcia et al, 2011;Passos et al, 2012). In this sciarid, the 5S ribosomal gene is localized in the autosomal A chromosome.…”