Inefficient fast PCR
AssigneeCaliper Technologies Corp.
InventorAnne R. Kopf-Sill
Life of this patent
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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.
Claims126
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.
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.
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.
An apparatus for amplifying one or more nucleic acids comprising,
- a body with at least one microscale channel fabricated therein;
- and
- a thermal control element coupled to the microscale channel, which control element cycles the temperature in one or more regions of the microscale channel at 4 second intervals or less.
An integrated system for amplifying one or more nucleic acids, comprising:
- an apparatus comprising a body with at least one microscale channel fabricated therein and a thermal control element coupled to the microscale channel;
- a computer and software for controlling one or more of:
- (i) temperature of at least one fluid within said microscale channel;
- (ii) number of different temperature-controlled zones within said microscale channel;
- and
- (iii) movement of at least one fluid present within said microscale channel.
Dependencies are inferred from the claim text.