Detection of target nucleic acid sequences by exonucleolytic activity using single-labeled immobilized probes on solid phase

A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a cyclic exonucleolytic reaction (CER) with no use of an upstream primer or an upstream probe on a solid phase, which comprises the steps of:

• (a) hybridizing the target nucleic acid sequence with an immobilized probe having a nucleotide sequence complementary to the target nucleic acid sequence and having a single label generating a detectable signal;
• wherein the immobilized probe is immobilized on a solid substrate through its 3′-end;
• (b) contacting the resultant of step (a) to a thermostable enzyme having a 5′ to 3′ exonuclease activity under conditions for cleavage of the immobilized probe;
• wherein the immobilized probe is digested by the thermostable enzyme having the 5′ to 3′ exonuclease activity to release the single label from the immobilized probe, resulting in decrease in a signal on the solid substrate;
• wherein the thermostable enzyme having the 5′ to 3′ exonuclease activity is a template-dependent nucleic acid polymerase having a 5′ to 3′ exonuclease activity;
• (c) denaturing the resultant of step (b);
• (d) repeating the steps (a)-(c) at least twice to further decrease the signal on the-solid substrate with no use of an upstream primer or an upstream probe;
• and
• (e) detecting the signal decrease on the solid substrate, such that the signal decrease by the cleavage of the immobilized probe is indicative of the presence of the target nucleic acid sequence;
• wherein the upstream primer or the upstream probe is a primer or a probe to be hybridized with a site downstream of a hybridized site of the immobilized probe.