PCR System for Real-time Quantification of Samples
This invention comprises a nucleic acid quantification kit and method for determining the initial concentration or mass fraction of a target nucleic acid present in a sample. More particularly, the invention relates to the use of real-time competitive quantitative polymerase chain reaction (PCR) and fluorescently labeled oligonucleotide probes to monitor the PCR reaction in real time. Also, an oligonucleotide probe can be used to obtain data through melting curve analysis. This system, therefore, provides a method for determining the copy number of a target nucleic acid present in a biological sample.
In one embodiment, the competitor nucleic acid sequence is prepared to have the identical sequence as the target sequence, except for a unique section located at an internal position on the competitive nucleic acid sequence. Compared to the target nucleic acid sequence, this unique section has the same overall nucleotide composition but a sequence that is sufficiently different to not cross-hybridize with a probe complementary to the target.
In another embodiment, the unique section of the competitor nucleic sequence may differ from the target sequence by only one base pair or may differ in length and/or composition.
To date, all real-time PCR quantification applications have been limited to quantification relative to an external standard curve. While this technique is very useful, it lacks control for tube-to-tube differences in PCR efficiency. This limitation of quantification with external standards has been addressed by competitive quantitative PCR methods. However, no real-time version is available. This invention differentiates target from competitor and includes gene quantification with the added control of an internal standard.
Stage of Development
European patent application 01 288 314 has been filed for this invention.
*Wittwer CT, Rasmussen RP et al. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 1997 Jan;22(1):130-1,134-8.
*Bennett CD, Rasmussen RP et al. The LightTyper: high-throughput genotyping using fluorescent melting curve analysis. Biotechniques 2003 Jun;34(6):1288-92, 1294-95.
Randy Rasmussen, David Eyre, Wade Stevenson, Brian Caplin, Deepika deSilva
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