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Nonradioactive method for quantitation of PCR products without hybridization with a specific probe.

A method is described for quantifying polymerase chain reaction (PCR) products by labeling them with biotinylated dUTP or dATP during the amplification step. The biotin-PCR products are size-separated by gel electrophoresis, transferred to a nylon membrane and then complexed with streptavidinalkaline phosphatase. The complexes are detected by reaction with a chemiluminescent substrate sheet. This detection takes advantage of the sensitivity of chemiluminesence, without requiring hybridization with a labeled probe. The yield of biotinylated PCR products has a linear dependence on the number of PCR cycles. Thus, this method can be used to quantify PCR products, including those made by reverse transcription (RT)-PCR. Simplicity, speed and sensitivity make this a useful method. RT-PCR is a powerful tool to quantify mRNA (1,5–7). The usual procedure involves RT of mRNA with random or poly(dT) primers, PCR amplification of cDNA with specific primers and then electrophoretic separation of the PCR products. Amplified cDNAs can be detected in gels with ethidium bromide staining, but this approach may not have the desired sensitivity. To increase sensitivity of detection and to prove identity of the products, PCR products are usually transferred from a gel to a membrane and hybridized with a labeled specific probe. Autoradiography, color reactions or chemiluminescence are used to visualize the labeled products. Detection methods that use chemiluminescence have sensitivities comparable to radioactive techniques (2–4, 8,9) and have the advantage of safety. In many experiments that quantify RT-PCR products, increased sensitivity of detection of the products is desired, but proof of their identity is unnecessary. In these cases, the target DNA sequence is known, the PCR primers yield a product of known size, and identity of the product has been previously confirmed by DNA sequencing or hybridization experiments. In such circumstances, the time and effort required by the hybridization step are spent only to increase sensitivity of detection. The method described is useful when increased sensitivity of detection of PCR products is desired, and proof of their identity is unnecessary. It is based on the ability of biotinylated nucleotides to be incorporated during PCR into DNA fragments up to 2600 bp in size (9,10). Incorporation of up to 75% of biotinylated nucleotides into PCR products can be done without significantly changing electrophoretical mobility of the products (10). Increased sensitivity is gained by use of chemiluminescence detection of the biotinylated PCR products. Time and effort are saved because there is no hybridization step. Biotin-21-dUTP and PCR primers for interleukin (IL)-1α, IL-1β, IL-2, IL3, IL-4, IL-5, IL-6, IL-8, IL-10, transforming growth factor-β (TGF-β), interferon-γ (IFN-γ) and β actin were purchased from CLONTECH Laboratories (Palo Alto, CA, USA). Biotin14-dATP was obtained from Life Technologies (Gaithersburg, MD, USA). Nytran nylon membranes, membraneblocking powder, streptavidin-alkaline phosphatase conjugate and chemiluminescent substrate sheets containing Lumi-Phos 530 were from the RadFree System (Schleicher & Schuell, Keene, NH, USA). Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by density gradient centrifugation using Histopaque-1077 (Sigma Chemical, St. Louis, MO, USA). Total cellular RNA was isolated from PBMC