Preparation of multi-locus DNA probe cocktail by liquid-phase reassociation.
We developed a simple, rapid method for the preparation of a DNA-fingerprinting probe cocktail, and tested its usefulness in paternity testing. Exploiting the property of tandemly repetitive DNA segments to be rapidly renatured after heat denaturation, we enriched restriction fragments of a child's genomic DNA for minisatellites by liquid phase reassociation followed by capture with immobilized streptavidin. We amplified and simultaneously labeled the reassociation product by anchored PCR using a digoxigenin-labeling mixture. Using this probe cocktail, we were able to detect fingerprints of paternity case trios, and the results were corroborated by DNA fingerprinting with a commercially available probe as well as by conventional phenotyping. Our method enables one to prepare a fresh cocktail of probes from the DNA sample under study during the overnight electrophoresis and Southern transfer steps in DNA fingerprinting, and eliminates the need of having an expensive probe of limited shelf life. If one has a practical outlook on DNA fingerprinting and regard it as a preliminary test, one does not have to use a cloned DNA probe. The present study demonstrates that a multi-locus probe cocktail serves such a practical purpose.
Tamaki
Y
Y
Department of Forensic Medicine, Oita Medical University, Japan.
Fukuda
M
M
Kishida
T
T
Wang
W
W
D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D015342:DNA Probes; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010334:Paternity; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D015342:DNA Probes