Method of automatically selecting oligonucleotide hybridization probes

A method of selecting oligonucleotide hybridization probes for detecting a mutation causing a mismatch in a DNA duplex, the method comprising the steps of:

• defining the mutation to be detected by
• a DNA sequence of a mutant DNA strand and
• a DNA sequence of a wild-type DNA strand corresponding to the mutant DNA strand;
• defining an integer variable L1 for a first length of the oligonucleotide hybridization probes from the place of mutation in a first direction;
• defining a range from m1 to n1 of possible values of the variable L1;
• defining an integer variable L2 for a second length of the oligonucleotide hybridization probes from the place of mutation in a second direction opposite to the first direction;
• defining a range from m2 to n2 of possible values of the variable L2;
• selecting at least one oligonucleotide hybridization probe from the group consisting of
• probes which are fully complementary to the mutant DNA strand,
• probes which are fully complementary to the wild-type DNA strand corresponing to the mutant DNA strand,
• probes which are fully complementary to an opposite sense mutant DNA strand which is the complementary DNA strand to the mutant DNA strand, and
• probes which are fully complementary to an opposite sense wild-type DNA strand which is the complementary DNA strand to the wild-type DNA strand corresponding to the mutant DNA strand;
• determining all possible hybridization probes from the possible values of the variables L1 and L2, and the selected probes;
• defining a thermodynamic nearest-neighbor model for calculating a melting point of a certain hybridization probe hybridized to a DNA strand, the melting point being a temperature at which a predetermined percentage of a multitude of identical pairs of said certain hybridization probe and said DNA strand is in an annealed state;
• calculating, on basis of the thermodynamic nearest-neighbor model, for all possible hybridization probes
• a first melting point of the respective probe hybridized with its complementary mutant DNA strand or opposite sense mutant DNA strand, respectively,
• a second melting point of the respective probe hybridized with its complementary wild-type DNA strand or opposite sense wild-type DNA strand, respectively, and
• a temperature difference between the second melting point and the first melting point;
• and
• ranking the possible hybridization probes with regard to the temperature difference.