Detection of quantitative polymerase chain reaction products by hybridization on magnetic support with 125I-radiolabeled probes: quantification of c-myc copy numbers.
We present a technique for determining c-myc copy numbers that can be used as a prognosis index for some cancers. The method is based on the use of both competitive polymerase chain reaction and hybridization of amplified products. Coamplification was performed directly on cells with a synthetic oligonucleotide used as internal standard. It recognized the same primer set as the target. Coamplified products were captured on streptavidin magnetic beads as solid support using a 5' biotinylated primer. DNA immobilized on this support was denatured with alkali. Each coamplified product (target and reference gene) was further hybridized to two distinct specific oligonucleotide probes. Gene amplification levels were determined using a standard curve obtained by serial dilutions of peripheral blood lymphocytes run along with the experimental samples. This approach provides a rapid (less than 2 days) and reproducible method for evaluating c-myc gene copy number and may be used to quantify any gene. Moreover, its format allows for automation.
Rhoer-Moja
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S
Laboratoire de Biophysique et d'Informatique Médical, UER de Médecine, Saint Etienne, France.
Cohen-Haguenauer
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O
Jouve
C
C
Healy
J C
JC
Vindimian
M
M
D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D015342:DNA Probes; D016259:Genes, myc / Q000235:genetics*; D006801:Humans; D007457:Iodine Radioisotopes; D008280:Magnetics; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D019809:Streptavidin; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D015342:DNA Probes; D007457:Iodine Radioisotopes; D001710:Biotin; D019809:Streptavidin