Patent ID: 8187813

Claim:
A method for amplifying a target nucleic acid sequence by polymerase chain reaction (PCR), comprising the steps of: (1) providing a tube-like container; (2) placing a PCR sample in the tube-like container, wherein the sample contains a template DNA having the target nucleic acid sequence to be amplified, DNA polymerases, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs), deoxyguanosine triphosphates (dGTPs), deoxythymidine triphosphates (dTTPs), and at least two oligonucleotide primers having a sequence complementary to the 3′ end of the target nucleic acid sequence wherein the primers are designed to have a melting temperature (T m ) between 40° C. to 90° C.; (3) embedding the bottom part of the container in a heat source, and then heating the PCR sample to allow the primers melt and maintaining a steady temperature in ° C. of the surface of the PCR sample (Ts) in the container less than the temperature in ° C. of the melting temperature of the primers (T m ) by at least 2° C., and controlling parameters of the PCR including total volume of the PCR sample (V), viscosity in Ns/m 2 of the PCR sample (u), inner diameter in mm of the container (d), and surface temperature in ° C. of the PCR sample in the container (Ts) so that a temperature gradient descending from the bottom to the top is resulted in the sample, which induces a convection and makes the following events occur sequentially and repeatedly in different regions of the PCR sample: (i) denaturation, in which the double-stranded template DNA separates into two single-stranded DNAs, (ii) annealing, in which the primers hybridize to the single-stranded DNAs, forming DNA-primer complexes, and (iii) polymerization, in which the primers in the DNA-primer complexes are extended by the DNA polymerase; wherein the parameters of the PCR: the total volume of the PCR sample (V), the viscosity in Ns/m 2 of the PCR sample (μ), the inner diameter in mm of the container (d), and the surface temperature in ° C. of the PCR sample in the container (Ts), are determined according to the formula below: V =( A×T s +B− 500μ+0.7)× e (1.86+100μ)d in which A is a value between −0.019 and −0.016, and B is a value between 1.85 and 2.27.