Method of increasing discrimination for single base mismatch using hurdle DNA and artificially mismatched probe

A method of detecting a polymorphism site in a target DNA having the polymorphic site, the method comprising:

• synthesizing a first probe by artificially substituting a base of a probe which is fully complementary to the test target DNA with a mismatched natural base such that the base on the polymorphic site of the test target DNA is fully complementary to its corresponding base in the first probe and the mismatched natural base in the first probe is not complementary to its corresponding base in the test target DNA;
• synthesizing a second probe by artificially substituting a base of another probe having a single base mismatch at the position corresponding to the polymorphic site of the target DNA with a mismatched natural base such that the base on the polymorphic site of the test target DNA is not complementary to its corresponding base in the second probe and the mismatched natural base in the second probe is not complementary to its corresponding base in the test target DNA, wherein the base of the another probe substituted by the mismatched natural base is not at the position corresponding to the polymorphic site of the target DNA;
• synthesizing a first hurdle DNA which is fully complementary to the first probe and is shorter than the first probe, wherein the melting temperature (Tm) of the hybrid of the first hurdle DNA and the first probe is lower than that of the hybrid of the target DNA and the first probe;
• synthesizing a second hurdle DNA which is fully complementary to the second probe and is shorter than the second probe, wherein the melting temperature (Tm) of the hybrid of the second hurdle DNA and the second probe is higher than that of the hybrid of the target DNA and the second probe;
• immobilizing the first and second probes on a substrate and forming immobilized first and second probes;
• hybridizing the immobilized first and second probes with the first and second hurdle DNAs, respectively;
• hybridizing the test target DNA with the immobilized first and second probes following the hybridizing the immobilized first and second probes with the first and second hurdle DNAs under conditions such that the first hurdle DNA is easily substituted by the target DNA and the second hurdle DNA is not easily substituted by the target DNA;
• determining the ratio of the relative amount of the test target DNA hybridized to the first probe to the relative amount of the test target DNA hybridized to the second probe thereby detecting the polymorphic site of the test target DNA based on the ratio of the relative amount of the test target DNA hybridized to the first probe to the relative amount of the test target DNA hybridized to the second probe.