Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean.
Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.
Pich
U
U
Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany.
Houben
A
A
Fuchs
J
J
Meister
A
A
Schubert
I
I
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D007887:Fabaceae / Q000235:genetics*; D005434:Flow Cytometry; D017343:Genes, Plant / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*
D017931:DNA Primers; D009841:Oligonucleotides; D004247:DNA