Nick translation reagent kit

Cloned satI, satII, satIII, and satIV DNA of M. gouazoubira were labelled with Orange- or Green-dUTP (Abbott, Abbott Park, IL, USA) using Nick Translation Reagent Kit (Abbott) to serve as probes for comparative FISH. FISH was performed using standard protocols [19 (link)]. Hybridization signals were examined using Zeiss Axio Imager.Z2 fluorescence microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) equipped with appropriate fluorescent filters and the Metafer Slide Scanning System (MetaSystems, Altlussheim, Germany). Images of well-spread metaphase cells were captured and analyzed using ISIS3 software (MetaSystems).

BAC DNAs were extracted and amplified by rolling circle amplification using the bacteriophage phi29 (Kit GE Healthcare, Amersham Biosciences, Buckinghamshire, UK). The quantity and purity of the amplified DNA are evaluated with Nanodrop (Thermo scientific) and a purification step is performed if necessary (DNA Clean and concentrator kit, Ozyme). The purified BACs are then conjugated to fluorochromes (Green-dUTP, Enzo life sciences; Aqua-dUTP, Enzo Life Sciences; Tetramethyl-Rhodamine-5-dUTP, Roche Diagnostics) with a Nick translation Reagent Kit (Abbott Laboratories). The double exonuclease and polymerase activity of the enzyme polymerase I removes the original nucleotides from the DNA matrix and incorporates the labeled deoxynucleotides. The labeled probes are then precipitated by addition of salts (5M NaCl) in an alcoholic medium to reach a high ionic strength and then precipitated at −80 °C for at least 2 h in the presence of human Cot-1 DNA (Invitrogen) to block non-specific hybridizations and suppress repetitive DNA sequences. The supernatant is removed after centrifugation for 20 min at 4 °C and 16,200× g, then the pellet is resuspended with hybridization buffer.

Metaphase spreads were obtained from phytohaemagglutinin-stimulated peripheral blood lymphocytes of the patient and both parents. Bacterial artificial chromosome (BAC) clones were selected from the Centro de Regulación Genómica Genome Browser [12 ]. Plasmid DNA was extracted from clones using QIAprep Spin Miniprep Kit (Qiagen) and labeled with Spectrum Orange dUTP (SpO) or Spectrum Green dUTP (SpG) using the Nick Translation Reagent Kit (Abbott Molecular Inc.). FISH experiments were carried out according to standard procedures. The slides were examined using a Nikon Eclipse E400 with appropriate filters for Spectrum Orange, Spectrum Green and the UV Filter for the DAPI nuclear counterstain. The signals were recorded with a CCD camera and processed by ISIS v5.1 fluorescence imaging system (MetaSystems).