Isothermal methods for amplifying nucleic acid samples
AssigneeTangen Biosciences, Inc.
InventorsJohn R. Nobile, John Davidson
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Abstract
The description provides two-stage methods of nucleic acid amplification and detection reactions, which are useful for rapid pathogen detection or disease diagnosis. In particular, the description provides a method comprising a first-stage slow rate amplification reaction followed by a plurality of second-stage fast rate amplification reactions that are simultaneously monitored in real-time, and wherein a rapid rate of amplification is indicative of the presence of a site of interest.
Claims24
A two-stage multiplexed nucleic acid amplification and real-time detection method comprising a. providing a composition comprising a target nucleic acid template and a combination of site-specific primers that anneal to the target nucleic acid template near a region of interest to be amplified;
- b. performing a first isothermal reaction to amplify the region of interest, thereby forming a primary amplicon;
- c. dividing (b) into at least two secondary reactions, and including in at least one of the reactions one or more site-specific secondary primer that is complementary to a site-specific primer binding site that may be present within the primary amplicon and defines a site of interest within the region of interest;
- d. performing a second isothermal reaction (second-stage reaction) thereby accelerating the amplification of the region of interest only if the site-specific primer binding site is complementary to the site-specific primer;
- and e. detecting and comparing the amplification rates of the at least two secondary reactions, wherein an enhanced relative rate of amplification in the reaction with the secondary primer indicates the presence of the site of interest that is complementary to the secondary primer.
Dependencies are inferred from the claim text.