Methods for rapid multiplexed amplification of target nucleic acids
First Claim
Patent Images
1. A method for simultaneously amplifying of a plurality of loci in a nucleic acid solution comprising:
- providing in single solutions contained in a plurality of reaction chambers located in a biochip, samples having at least ten and up to 250 target nucleic acid loci to be amplified, with at least ten and up to 250 different primer pairs, each primer pair hybridizing to one of the at least ten and up to 250 loci to be amplified, said solution further comprising;
(i) one or more buffers;
(ii) one or more salts;
(iii) a nucleic acid polymerase; and
(iv) nucleotides; and
providing a TCE, comprising a means for heating and cooling, at least one thermosensor, a controller that receives signals from said at least one thermosensor and a power supply, said at least one thermosensor positioned and configured to measure the effective temperature of each of the single solutions in the reaction chambers of the biochip and to provide feedback to the TCE to heat or cool the solution to set or maintain the solution at a desired temperature;
simultaneously amplifying said plurality of loci by using the at least one thermosensor, the controller and the TCE to sequentially thermally cycle the temperature of the nucleic acid solution in each reaction chamber between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles at heating and cooling rates of about 4-150°
C./sec, and minimizing the transition times between the extension, denaturation, and annealing states to yield a plurality of amplified loci in each reaction chamber in 97 minutes or less.
5 Assignments
0 Petitions
Accused Products
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.
-
Citations
28 Claims
-
1. A method for simultaneously amplifying of a plurality of loci in a nucleic acid solution comprising:
-
providing in single solutions contained in a plurality of reaction chambers located in a biochip, samples having at least ten and up to 250 target nucleic acid loci to be amplified, with at least ten and up to 250 different primer pairs, each primer pair hybridizing to one of the at least ten and up to 250 loci to be amplified, said solution further comprising; (i) one or more buffers; (ii) one or more salts; (iii) a nucleic acid polymerase; and (iv) nucleotides; and providing a TCE, comprising a means for heating and cooling, at least one thermosensor, a controller that receives signals from said at least one thermosensor and a power supply, said at least one thermosensor positioned and configured to measure the effective temperature of each of the single solutions in the reaction chambers of the biochip and to provide feedback to the TCE to heat or cool the solution to set or maintain the solution at a desired temperature; simultaneously amplifying said plurality of loci by using the at least one thermosensor, the controller and the TCE to sequentially thermally cycle the temperature of the nucleic acid solution in each reaction chamber between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles at heating and cooling rates of about 4-150°
C./sec, and minimizing the transition times between the extension, denaturation, and annealing states to yield a plurality of amplified loci in each reaction chamber in 97 minutes or less. - View Dependent Claims (2, 4, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
-
-
3. A method for simultaneously amplifying 5 or more loci in a nucleic acid solution comprising:
providing in single solutions contained in a plurality of reaction chambers located in a biochip, samples having at least five target nucleic acid loci to be amplified, with at least five different primer pairs, each primer pair hybridizing to one of at least five loci, said solution further comprising; (i) one or more buffers; (ii) one or more salts; (iii) a nucleic acid polymerase; and (iv) nucleotides; and providing a TCE, comprising a means for heating and cooling, at least one thermosensor, a controller that receives signals from said at least one thermosensor and a power supply, said at least one thermosensor positioned and configured to measure the effective temperature of each of the single solutions in the reaction chambers of the biochip and to provide feedback to the TCE to heat or cool the solution to set or maintain the solution at a desired temperature; simultaneously amplifying said plurality of loci by using the at least one thermosensor, the controller and the TCE to sequentially thermally cycle the temperature of the nucleic acid solution in each reaction chamber between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles at heating and cooling rates of about 4-1 50°
C./sec, and minimizing the transition times between the extension, denaturation, and annealing states to yield 5 or more amplified loci in each reaction chamber in 97 minutes or less.- View Dependent Claims (6, 7, 8)
Specification