PROCESS FOR DETECTING POINT MUTATIONS IN WHICH THE PRIMER IS BOUND TO A SOLID SUPPORT AND EXTENSION DOES NOT OCCUR IF THE TERMINAL NUCLEOTIDE(S) MIS-MATCH

摘要

A process for determination of at least one target nucleotide at at least one particular location within at least one polynucleotide sequence in a test sample, said target nucleotide(s) constituting a 5' terminal nucleotide of a target nucleotide sequence within said polynucleotide sequence(s), said process including the steps of: (i) immobilizing one or more arrays of identical oligonucleotide primers with a solid support defining one or more reaction zones wherein each of said oligonucleotide primers in a respective array is capable of hybridizing with a corresponding target nucleotide sequence; (ii) contacting the reaction zone with the test sample; (iii) hybridizing at least one of said oligonucleotide primers with the polynucleotide sequence to form a hybrid wherein the polynucleotide sequence extends in a 3' to 5' direction beyond the 3' terminal nucleotide of the at least one oligonucleotide primer; (iv) extending the at least one oligonucleotide primer of the hybrid beyond the 3' terminal nucleotide thereof in the 5' to 3' direction using the polynucleotide sequence as a template in the case when the 3' terminal nucleotide of the at least one oligonucleotide primer is complementary to the 5' terminal nucleotide of said target nucleotide sequence, said extension effected in the presence of a polymerisation agent and nucleotide precursors to form a duplex including an extended primer molecule wherein at least one nucleotide incorporated into the extended primer molecule is a detectably-modified nucleotide; (v) denaturing the duplex to free the polynucleotide sequence from the extended primer molecule; (vi) carrying out steps (iii) to (v) one or more times; and (vii) detecting presence of a signal associated with the detectable-modified nucleotide in the extended primer molecule at the reaction zone to effect said determination. And an apparatus for PCR.