Methods for rapid multiplexed amplification of target nucleic acids
Inventors
Selden, Richard F. • Tan, Eugene • Lam, Heung Chuan • Giese, Heidi Susanne • Kellogg, Gregory John • Wright, John A.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9494519-B2
Publication Date
2016-11-15
Expiration Date
Abstract
A fast, multiplexed PCR system is described that can rapidly generate amplified nucleic acid products, for example, a full STR profile, from a target nucleic acid. Such systems include, for example, microfluidic biochips and a custom built thermal cycler, which are also described. The resulting STR profiles can satisfy forensic guidelines for signal strength, inter-loci peak height balance, heterozygous peak height ratio, incomplete non-template nucleotide addition, and stutter.
Core Innovation
The disclosure describes rapid multiplex nucleic-acid amplification in reaction chambers of a microfluidic biochip using a thermosensor-feedback thermal cycler. At least one thermosensor measures the effective temperature of the solutions, and feedback controls heating and cooling to maintain target temperatures during thermal cycling. This process minimizes transitions among denaturing, annealing, and extension states to produce amplified loci across the reaction chambers within a stated time limit.
The system targets rapid multiplex amplification across ranges spanning at least five loci and up to 250 loci. Reported experimental characterizations include full forensic STR profiles generated across biochip reaction chambers, electropherograms, and sequence-trace data. In addition, the disclosure evaluates starting DNA template levels against resulting signal strength, peak height ratio, non-template addition, and stutter measurements across the biochip chambers.
Claims Coverage
The claims cover simultaneous amplification of multiple nucleic acid loci in biochip reaction chambers across three inventive features. The independent claims utilize thermosensor feedback to control thermal cycling and differ in the claimed scope of target loci.
Simultaneous multiplex amplification across target loci
The claims provide for simultaneously amplifying multiple target nucleic acid loci, spanning at least five loci or 10 to 250 loci, in solutions held within biochip reaction chambers using primer pairs directed to the target loci.
Thermosensor-feedback temperature control
At least one thermosensor measures the effective temperature of the solutions in the biochip reaction chambers and provides feedback to a temperature control element that sets or maintains desired solution temperatures during thermal cycling.
Rapid thermal cycling with minimized state transitions
The method cycles reaction solutions through denaturing, annealing, and extension states while minimizing thermal transitions between states, yielding amplified loci within a stated time limit.
The independent claims combine multiplex amplification in biochip reaction chambers with thermosensor-feedback temperature control and minimized thermal transitions, differing primarily in the specified number of target loci.
Stated Advantages
Minimizing thermal-transition times enables amplified loci to be produced within the stated time limit.
Documented Applications
Forensic STR profiling, including analysis of amplified nucleic acid products in accordance with forensic interpretation guidelines.
Interested in licensing this patent?