Primer-induced labeling of pea and field bean chromosomes in situ and in suspension
Language English Country England, Great Britain Media print
Document type Research Support, Non-U.S. Gov't, Technical Report, Journal Article
PubMed
7495553
Knihovny.cz E-resources
- MeSH
- Deoxyuracil Nucleotides MeSH
- DNA Primers * MeSH
- DNA, Plant analysis MeSH
- DNA Probes MeSH
- Fabaceae genetics MeSH
- Fluorescein MeSH
- Fluoresceins MeSH
- Microscopy, Fluorescence MeSH
- In Situ Hybridization MeSH
- Plants, Medicinal * MeSH
- Molecular Sequence Data MeSH
- Nucleolus Organizer Region MeSH
- Polymerase Chain Reaction MeSH
- Repetitive Sequences, Nucleic Acid MeSH
- Base Sequence MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Technical Report MeSH
- Names of Substances
- Deoxyuracil Nucleotides MeSH
- deoxyuridine triphosphate MeSH Browser
- DNA Primers * MeSH
- DNA, Plant MeSH
- DNA Probes MeSH
- Fluorescein MeSH
- Fluoresceins MeSH
A protocol for primed in situ DNA labeling (PRINS) was optimized for pea (Pisum sativum L.) and field bean (Vicia faba L.) chromosomes attached to coverslips. Cloned DNA or synthetic oligonucleotides were used as probes for repetitive DNA sequences (rDNA, Fok-element) and different reaction conditions were tested to achieve the highest specific signal-to-background ratio. A procedure based on direct labeling by fluorescein-dUTP was compared with an indirect one using digoxigenin detected by fluorescently labeled antibody. Under optimal conditions, strong and specific signals were obtained exclusively on chromosome regions known to contain respective DNA sequences. Compared to the direct labeling, significantly stronger signals were obtained when the indirect procedure was used. Both types of labeling were successfully applied to chromosomes in suspension and were shown to produce signals comparable to that obtained with chromosomes attached to coverslips. It is expected that primed in situ DNA labeling en suspension (PRINSES) will provide a basis for flow-cytometric discrimination and sorting of otherwise indistinguishable chromosomes according to their specific fluorescent labeling.
Chromosome analysis and sorting
Chromosomes in the flow to simplify genome analysis
Chromosome-based genomics in the cereals
Flow cytogenetics and plant genome mapping
Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.)
GENBANK
X00801, X16614
