Method to characterize nucleic acid

A method for detecting nucleic acid hybridization, comprising the steps of:

• a) measuring the energy (E0) of a nucleic acid probe having a sequence identity of SEQ ID NOs:
• 1 or 6 in a buffer, comprising the steps of
• a1) providing a buffer medium, wherein the buffer preferably comprises 50 mM Tris-HCl, 22 mM EDTA and 1.2% Triton X-100, where the pH is 7.0-7.5;
• a2) providing the nucleic acid probe with a fluorescence-label within that buffer medium;
• a3) measuring the energy (E0) of that system by exciting the fluorescence-tag and measuring the fluorescence of the system;
• b) measuring the energy (EK) of the probe hybridized to a control nucleic acid having a sequence identity of SEQ ID NOs:
• 2 or 7, comprising the steps of:
• b1) using the same buffer medium of step a);
• b2) providing the probe of step a2) and the control nucleic acid molecule which is 100% complementary to the nucleic acid probe;
• b3) letting the nucleic acid probe and the complementary nucleic acid molecule hybridize at a temperature Ta which is from 0° C. to the melting temperature of both molecules, preferably from above 0° C. and up to 5° C. below the melting temperature of both molecules;
• b4) measuring the energy (EK) of that system by exciting the fluorescence-tag and measuring the fluorescence of the system;
• c) measuring the energy (ES) of the probe hybridized to a sample nucleic acid, having a sequence identity of SEQ ID Nos:
• 3 or 8, comprising the steps of:
• c1) using the same buffer medium of steps a) and b);
• c2) providing the nucleic acid probe of steps a2) and b2) and the sample nucleic acid molecule;
• c3) letting the nucleic acid probe and the sample nucleic acid molecule hybridize at the same temperature Ta as used in step b3);
• c4) measuring the energy (ES) of that system by exciting the fluorescence-tag and measuring the fluorescence of the system;
• and optionally,
• d) repeating steps a)-c) in a buffer with a chaotropic agent;
• thereby receiving the energies E′0, E′K and E′S.
• e) calculate the energy-shifts, comprising the steps of:
• e1) calculate ΔEH according to the formula ΔEH=E0−EK;
• e2) calculate ΔEHM according to the formula ΔEHM=E0−ES;
• e3) calculate ΔE′H according to the formula ΔE′H=E′0−E′K;
• e4) calculate ΔE′HM according to the formula ΔE′HM=E′0−E′S;
• e5) calculate ΔΔE according to the formula ΔΔE=|ΔEH|−|ΔEHM|;
• optionally, calculate ΔΔE′
• e6) optionally, calculate ΔΔE′ according to the formula ΔΔE′=|ΔE′HM|−|ΔE′H;
• wherein |ΔEH|>|ΔEHM|—means that there is at least a single mispaired base-pair in the duplex of probe and sample nucleic acid;
• wherein ΔΔE means a decrease of the duplex melting temperature;
• wherein, optionally, |ΔE′H|<|ΔE′HM| means that in the duplex are mispaired base-pairs and
• wherein ΔΔE′ can be used to calculate the number of mispaired base-pairs by using the formula ΔΔE′˜n(Tm−Ta)ΔS, where n is the number of mispaired base pairs. Tm− is the melting temperature, Ta is the same temperature as of step b3), and ΔS is the entropy of a single broken hydrogen bond.