Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopicin situhybridization

Abstract

A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization.

Human μ opiate receptor

“…6,7 or X) chromosome. To determine the specific chromosome and band, cells were G-banded by fluorescence plus Giemsa [31] techniques, and photographs of banding patterns aligned with photographs of the fluorescence in situ hybridization signals to determine sub-band location. Eighteen signals of 27 analyzable signals (67%) were on chromosome 6, bands q24-25 (Fig.…”

“…A genomic clone, pHG4, was nick-translated with biotin-14 dATP (BRL, Gaithersburg, MD), with 81% incorporation as determined by tritium tracer incorporation. Slides with chromosome spreads were made from normal male lymphocytes cultured with BrdU [31]. Fluorescence in situ hybridization was performed as described [32] with modifications.…”

A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs

“…phSVMT1 was nick-translated with biotin-14 dATP (BRL, Gaithersburg, MD), with 2 1% incorporation as determined by tritium tracer incorporatton. Slides with chromosome spreads were made from normal male lymphocytes cultured with BrdU [16]. Fluorescence in situ was performed as described [17] with modifications.…”

“…g-12) chromosome. To determine the specific chromosome and band, cells were G-banded by fluorescence plus Giemsa [16] techniques, and photographs of VESICLE LUMEN CVX?PLASM banding patterns aligned with photographs of the fluorescence in situ hybridization signals to determine subband location. 26 of 30 analyzable metaphases (87%) were on chromosome 10, largely on band lOq25 (Fig.…”

Origin of marrow stromal cells and haemopoietic chimaerism following bone marrow transplantation determined by in situ hybridisation

“…Two methods were used to detect hybridised biotinylated probes. In the first, the hybridised biotinylated probes were detected by an avidin-alkaline phosphatase conjugate (Dako, UK) as described The second method used a rabbit anti-biotin polyclonal antibody and an immunoperoxidase diaminobenzidine (DAB)-silver reaction to detect the biotinylated probe (Bhatt et al, 1988). The slides were first washed in PBS then immersed in a 3% solution of hydrogen peroxide (Gibco) in industrial methylated spirits for 30 min to block endogenous peroxide activity.…”