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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.

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