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Quantitative Polymerase Chain Reaction Using a DNA Hybridization Assay Based on Surface-Activated Microplates

1995; Elsevier BV; Volume: 225; Issue: 2 Linguagem: Inglês

10.1006/abio.1995.1150

1096-0309

Carsten Berndt, M. Bebenroth, K. Oehlschlegel, Falk Hiepe, W Schössler,

Molecular Biology Techniques and Applications

A method for DNA quantification on microplates based on the hybridization between single-stranded target and solid-phase bound capture DNA is presented. Binding of the capture DNA to the microplates was attained by surface activation using organosilanes. Detection of hybridized DNA was performed by an enzyme-linked assay taking advantage of the labeled target DNA. Basic test characteristics are described and application examples are given demonstrating its feasibility for a quantitative PCR (QPCR). The QPCR was carried out by coamplifying an immunoglobuline complementarity determining region 3 (CDR3)-coding plasmid DNA with a synthetic internal standard (IS). IS and plasmid CDR3 both shared primer binding sites and produced length-identical amplicons. The amplified DNA was quantified following differential hybridization with IS- and plasmid-specific capture probes. Based on the product ratios of plasmid to IS, the initial amounts of plasmid DNA were calculated. This way, QPCR was shown to be capable of detecting initial concentration differences of plasmid DNA of about 30%.