Chemical and thermal assisted nucleic acid amplification methods
Disclosed herein, inter alia, are novel methods pertaining to nucleic acid amplification and detection. Devices, compositions, and kits for use in such methods are also provided.
1 . A method of amplifying a template polynucleotide, said method comprising:
(a) annealing a template polynucleotide to a first immobilized primer on a solid support at a first temperature, wherein the first immobilized primer is complementary to a sequence of the template polynucleotide, wherein the first temperature is about 25° C. to about 45° C.;
(b) contacting the solid support with an extension solution and extending the first immobilized primer with a polymerase to generate a complement template polynucleotide, wherein contacting the solid support with an extension solution occurs at the first temperature and remains in contact with the solid support as the temperature is increased to a second temperature;
(c) contacting the solid support with a chemical denaturant and separating the complement template polynucleotide from the template polynucleotide, wherein:
i) contacting the solid support with a chemical denaturant occurs at the second temperature and remains in contact with the solid support as the temperature is decreased to the first temperature, and
ii) the second temperature is greater than the first temperature by 12° C. to 18° C.;
(d) removing the chemical denaturant and annealing the complement template polynucleotide to a second immobilized primer on said solid support at the first temperature, wherein the second immobilized primer is complementary to a sequence of the complement template polynucleotide, and;
(e) contacting the solid support with the extension solution and extending the second immobilized primer with the polymerase to generate an amplification product.
2 . The method of claim 1 , further comprising extending the first immobilized primer at the first temperature.
3 . The method of claim 1 , further comprising extending the first immobilized primer at the second temperature.
4 . The method of claim 1 , wherein step (c) comprises decreasing the second temperature to the first temperature occurs at a controlled rate.
5 . The method of claim 1 , wherein step (b) comprises increasing the first temperature to the second temperature at a controlled rate.
6 . The method of claim 1 , wherein the first temperature is about 40° C. to about 45° C.
7 . The method of claim 1 , wherein the second temperature is about 55° C. to about 62° C.
8 . The method of claim 1 , further comprising prior to step (a) contacting the solid support with a sample comprising a template polynucleotide.
9 . The method of claim 1 , further comprising repeating steps (a) to (e), thereby amplifying the template polynucleotide.
10 . The method of claim 1 , wherein the chemical denaturant comprises formamide, ethylene glycol, or sodium hydroxide.
11 . The method of claim 1 , wherein removing the chemical denaturant comprises application of a wash solution.
12 . The method of claim 1 , wherein the first temperature is about 40° C. and the second temperature is about 58° C.
13 . The method of claim 1 , in step (b), the solid support remains in contact with the extension solution for 45 seconds at the second temperature, where the second temperature is 58° C.
14 . The method of claim 1 , in step (c), the solid support remains in contact with the chemical denaturant for 45 seconds as the temperature reduces to the first temperature, where the first temperature is 42° C.
15 . The method of claim 1 , in step (c), the solid support remains in contact with the chemical denaturant for 5-15 seconds at the second temperature prior to decreasing to the first temperature.
16 . A method of amplifying a template polynucleotide, said method comprising:
(a) annealing a template polynucleotide to a first immobilized primer on a solid support at a first temperature, wherein the first immobilized primer is complementary to a sequence of the template polynucleotide, wherein the first temperature is about 35° C. to about 45° C.;
(b) contacting the solid support with an extension solution and extending the first immobilized primer to generate a complement template polynucleotide, wherein contacting the solid support with an extension solution occurs at the first temperature and remains in contact with the solid support as the temperature is increased to a second temperature;
(c) contacting the solid support with a chemical denaturant and separating the complement template polynucleotide from the template polynucleotide, wherein:
i) contacting the solid support with a chemical denaturant occurs at the second temperature and remains in contact with the solid support as the temperature is decreased to the first temperature, and
ii) the second temperature is 12° C. to 18° C. greater than the first temperature;
(d) removing the chemical denaturant and annealing a complement template polynucleotide to a second immobilized primer on said solid support at the first temperature, wherein the second immobilized primer is complementary to a sequence of the complement template polynucleotide;
(e) contacting the solid support with the extension solution and extending the second immobilized primer with the polymerase to generate an amplification product.
17 . The method of claim 16 , wherein the first temperature and the second temperature differ by about 15° C.
18 . The method of claim 16 , wherein the first temperature is about 40° C. to about 45° C.
19 . The method of claim 16 , wherein the second temperature is about 55° C. to about 62° C.
20 . The method of claim 16 , in step (b), the solid support remains in contact with the extension solution for 45 seconds at the second temperature, wherein the second temperature is 58° C.
21 . The method of claim 16 , in step (c), the solid support remains in contact with the chemical denaturant for 45 seconds as the temperature reduces to the first temperature, where the first temperature is 42° C.
22 . The method of claim 16 , in step (c), the solid support remains in contact with the chemical denaturant for 5-15 seconds at the second temperature prior to decreasing to the first temperature.