Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization.
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.
Bhatt
B
B
University of Oxford, Nuffield Department of Pathology, John Radcliffe Hospital, UK.
Burns
J
J
Flannery
D
D
McGee
J O
JO
D002478:Cells, Cultured; D002871:Chromosome Banding; D002877:Chromosomes, Human / Q000648:ultrastructure*; D005796:Genes; D006801:Humans; D007621:Karyotyping; D008214:Lymphocytes / Q000166:cytology; D008677:Metaphase; D008938:Mitosis; D009693:Nucleic Acid Hybridization