High efficiency of electron microscopy hybridization (EMISH) techniques: Comparison of methods and applications

Journal Article · Thu Sep 01 04:00:00 EDT 1994 · American Journal of Human Genetics

OSTI ID:134471

Scott, P; Fetni, R; Lemieux, N ^[1]

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1. Universite de Montreal, Quebec (Canada); and others

Methods and applications of transmission electron microscopy, for the study of surface-spread metaphases after in situ hybridization, are described. The electron microscope is able to detect single and repetitive DNA sequences at high spatial resolution. Comparison between fluorescent and electron microscope in situ hybridization signals was made using different types of sequences, such as telomeres, {alpha}-satellite DNAs, large and small single-copy sequences. In this study, the probes were disclosed with gold particles of different sizes while the chromosome bands were simultaneously revealed with a high concentration of antibodies against the BrdUrd-substituted DNA. Suppression strategies have been used to reveal hybridization of unique sequences. Different probe modification techniques were tested for digoxigenin, biotin, BrdUrd, fluorescein, etc. Using two closely located sequences within the RB1 gene, it was possible to observe two distinct spots in EMISH while only one oblong signal was visible in fluorescence. Even when two sequences appear intermingled or overlapping in fluorescence (intermediate color), the characteristically different signals in EMISH (distinct sizes of gold particles) allowed discrimination of the true limits of each sequence on the metaphase or interphase chromosome. The improved resolution obtained with electron microscopy in situ hybridization opens up great possibilities for high resolution localization of small DNA sequences, lateral positioning of sequences, size quantification and chromatin organization.

• OSTI ID: 134471
• Report Number(s): CONF-941009--
• Journal Information: American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
• Country of Publication: United States
• Language: English

Related Subjects

44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
55 BIOLOGY AND MEDICINE
BASIC STUDIES
AMINO ACID SEQUENCE
BANDING TECHNIQUES
CHROMATIN
DETECTION
DNA
DNA HYBRIDIZATION
DNA SEQUENCING
EFFICIENCY
FLUORESCENCE
GENES
GENETIC MAPPING
HUMAN CHROMOSOMES
MITOSIS
PROBES
RESOLUTION
SIZE
TELOMERES
TRANSMISSION ELECTRON MICROSCOPY