Inefficient fast PCR
First Claim
Patent Images
1. A method of performing a cyclic polymerase-mediated reaction, the method comprising:
- incubating a polymerase, a plurality of template molecules, and a plurality of primer molecules for a first time (t1), wherein t1≦
0.5(t2), wherein t2 is the time required for the duplication of at least 90% of the plurality of template molecules when said primer molecules and said polymerase are present in non-rate limiting amounts; and
repeating the incubation of said polymerase, said plurality of template molecules, and said plurality of primer molecules, for time t1, for a number of cycles, x1, wherein x1 is greater than the number of cycles required to produce a detectable amount of product when said polymerase, said plurality of template molecules, and said plurality of primer molecules are incubated for time t2, wherein the total amount of time required to generate a detectable amount of product using said increased number of said reduced-time cycles is less than the total amount of time required to generate a detectable amount of product sing the standard number of full-time cycles.
1 Assignment
0 Petitions
Accused Products
Abstract
Methods of performing fast polymerase mediated reactions are provided. These reactions can be used in an inefficient fashion in the cycles of the polymerase mediated reactions to produce product at a much faster rate than conventional polymerase mediated reaction methods. Integrated systems for performing these methods are also provided.
340 Citations
104 Claims
-
1. A method of performing a cyclic polymerase-mediated reaction, the method comprising:
-
incubating a polymerase, a plurality of template molecules, and a plurality of primer molecules for a first time (t1), wherein t1≦
0.5(t2), wherein t2 is the time required for the duplication of at least 90% of the plurality of template molecules when said primer molecules and said polymerase are present in non-rate limiting amounts; and
repeating the incubation of said polymerase, said plurality of template molecules, and said plurality of primer molecules, for time t1, for a number of cycles, x1, wherein x1 is greater than the number of cycles required to produce a detectable amount of product when said polymerase, said plurality of template molecules, and said plurality of primer molecules are incubated for time t2, wherein the total amount of time required to generate a detectable amount of product using said increased number of said reduced-time cycles is less than the total amount of time required to generate a detectable amount of product sing the standard number of full-time cycles. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
-
-
34. A method of performing a PCR, comprising:
-
(i) performing the PCR using a reduced-time cycle, which reduced-time cycle is shorter in duration than a full-time cycle, which full-time cycle is sufficient in duration for the duplication of at least 90% of the templates present in said full-time cycle; and
(ii) using an increased number of said reduced-time cycles compared to a standard number of full-time cycles, which standard number of full-time cycles is sufficient to generate a detectable amount of product, wherein the total amount of time required to generate a detectable amount of product using said increased number of said reduced-time cycles is less than the total amount of time required to generate a detectable amount of product using the standard number of full-time cycles. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70)
-
-
71. A method of amplifying a template, the method comprising incubating the template with at least one complementary primer and a polymerase, wherein the incubation comprises:
-
(i) denaturing the template to provide a single stranded template;
(ii) hybridizing the at least one complementary primer and the single stranded template; and
(iii) extending the resulting hybridized primer with the polymerase;
wherein the total time for the denaturing, hybridizing, and extending is about 6 seconds or less; and
(iv) repeating steps (i), (ii), and (iii) until a detectable amount of product is produced, wherein the total amount of time required to generate a detectable amount of product is less than the total amount of time required to generate a detectable amount of product using the standard number of full-time cycles. - View Dependent Claims (72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104)
-
Specification