Description of Invention:
This invention is in the field of multiplex real-time polymerase chain reaction (PCR). In particular, the invention pertains to the quantification of multiple amplicons in a single polymerase chain reaction based on the different melting temperatures of amplicons.
PCR is a primer-directed in vitro reaction for the enzymatic amplification of a fragment of DNA, involving repetitive cycles of DNA template denaturation, primer annealing to the DNA template, and primer extension. The result is an exponential accumulation of a specific DNA fragment or amplicon from an initial nominal amount of sample DNA templates. Multiplex PCR offers a more efficient approach to PCR, whereby multiple pairs of primers are used to simultaneously amplify multiple amplicons in a single PCR reaction. The simultaneous amplification of various amplicons decreases both the cost and turn-around time of PCR analysis, minimizes experimental variations and the risk of cross-contamination, and increases the reliability of end results. Multiplex PCR has gained popularity in many areas of DNA testing, including prognosis, diagnostic, gene deletion analysis, mutation and polymorphism analysis, genotyping and DNA array analysis, RNA detection, pharmacogenomics and identification of microorganisms.
Real-time PCR has been developed to overcome limitations in quantifying amplicons during an ongoing PCR reaction, since traditional PCR and multiplex PCR are often limited to a qualitative analysis of end-product amplicons. Real-time PCR is based on the principles that emission of fluorescence from dyes directly or indirectly associated with the formation of newly synthesized amplicons or the annealing of primers with DNA templates can be detected and is proportional to the amount of amplicons in each PCR cycle. The resulting emission curve can then be used to calculate the initial copy number of a nucleic acid template at the beginning of the PCR reaction. Real-time PCR eliminates the need for post PCR steps and is highly recognized for its high sensitivity, precision and reproducibility. This invention is directed to methods for real-time monitoring and quantification of multiple amplicons in a single multiplex real-time PCR reaction based on the use of a double stranded DNA dye and the melting temperature discrepancy among the amplicons.
Inventors:
Enrique Zudaire Ubani (NCI) Frank Cuttitta (NCI)
Portfolios: Infectious Diseases Devices/Instrumentation Cancer
Cancer -Diagnostics-In Vitro-DNA Based Infectious Diseases -Diagnostics-Viral-Non-AIDS (only) Cancer -Diagnostics Devices/Instrumentation-Diagnostics Infectious Diseases -Diagnostics
For Additional Information Please Contact: Susan Ano Ph.D.
NIH Office of Technology Transfer
6011 Executive Blvd, Suite 325
Rockville, MD 20852-3804
Phone: (301) 435-5515
Email: anos@mail.nih.gov
Fax: (301) 402-0220
