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CGH labeling protocol for small volumes of DNA sample

Eléonore Eymard-Pierre, Cytogenetics Laboratory, CHU Clermont-Ferrand, Clermont-Ferrand, France
Morgan Mathieu, Enzo Life Sciences, Lausen, Switzerland
Wini Luty, Enzo Life Sciences, Farmingdale, USA


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Introduction

Recent advances in genomics have dramatically increased our capacity to analyze both normal and cancer cells, revealing a multitude of changes in genomic DNA, such as mutations and copy number alterations. Array comparative genomic hybridization (aCGH) has become a valuable, genome-wide screening tool for the detection of chromosomal aberrations in the form of copy number variations. The technique is relatively simple involving fluorescent labeling of sample DNA followed by co-hybridization with normal control DNA spotted onto a glass slide or a chip. The major advantage of aCGH is that it offers improved resolution compared to traditional techniques and is more robust than mRNA expression arrays. Recent studies demonstrated the ability of aCGH to detect submicroscopic chromosomal imbalances in the range of a few megabases in 10–15% of patients with mental retardation and multiple congenital defects. Consequently, an increasing number of cytogenetic facilities have implemented this technique not only for postnatal and cancer diganosis but also for prenatal diagnosis. Since a simple DNA sample is required, much of the laborious steps in traditional karyotyping and cell culture can be circumvented.

The cytogenetics laboratory of the CHU Clermont-Ferrand (France) conducts on a daily basis the cytogenetic analysis of prenatal (e.g. chromosome number variations), postnatal (e.g. gene fusion, mutations) and oncological/hematological (e.g. gene fusion, gene expression and mutations) samples. The laboratory has at its disposal a range of chromosomal and molecular techniques to deliver cytogenetic diagnostics to the whole Auvergne region. Amongst the services offered by this laboratory is included an array CGH platform. Engineers and research technicians working on this platform rely on small amounts of genomic material, which are extracted, purified and eluted in small volumes to carry out subsequent experiments with concentrated DNA samples. The main objective of this study was to look at the compatibility of such a sample type with the CGH labeling kit from Enzo Life Sciences. Upon adjustment of the volumes of the different reagents, satisfactory yield, high specific activity, high signal intensity and low derivative log ratios were achieved with CGH labeling kit from Enzo Life Sciences whilst demonstrating significantly improved labeling efficiency when compared with Agilent’s SureTag® DNA labeling kit.

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