Methods using exogenous, internal controls and analogue blocks d

Details Methods using exogenous, internal controls and analogue blocks d Methods using exogenous, internal controls and analogue blocks d

1998-03-26

1999-09-14

Horlick, Kenneth R.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 6, 435 911, 536 2431, 536 2432, 536 2433, C12P 1934, C12Q 168, C07H 2104

Patent

active

059522020

ABSTRACT:
Reporter-quencher probe assays of nucleic acid amplification, such as PCR, are rendered more meaningful by the addition of internal control reagents. An internal control polynucleotide is amplified with internal control primers and the product is measured by correlation with increased fluorescence by polymerase mediated-exonuclease cleavage or hybridization of the internal control probe. Probes specific for target and internal control polynucleotides are labelled with spectrally resolvable reporters, allowing for concurrent detection and measurement of target and control amplification. A kit of all PCR reagents can be dispensed into reaction chambers in a high-throughput system for rapid and accurate nucleic acid amplification assay, with real-time or end-point measurements. Fluorescent signals correlated to target and internal control levels are spectrally resolvable and measured concurrently. A non-extending oligonucleotide or nucleic analog "block", complementary to the internal control polynucleotide, is added to the amplification mixture to preclude amplification of the internal control polynucleotide and function as an internal negative control. The amplification control reagents, kits, and methods of the present invention provide positive and negative control tests occurring within, and measurable within, the reaction chamber.

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