Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification.
We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.
Biomedical Sciences Division, Lawrence Livermore National Laboratory, California 94550.
H U
HU
Weier
R
R
Segraves
D
D
Pinkel
J W
JW
Gray
D001710:Biotin / Q000031:analogs & derivatives; D004247:DNA / Q000032:analysis / Q000096:biosynthesis*; D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes / Q000032:analysis; D005784:Gene Amplification; D006801:Humans; D008297:Male; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D014998:Y Chromosome / Q000032:analysis*
D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D001710:Biotin; C045931:biotin-11-dUTP; D004247:DNA