Detection of target nucleic acid sequences by cyclic exonucleolytic reactions
The present invention relates to the detection of a target nucleic acid sequence by a cyclic exonucleolytic reaction. The present method enabling to generate signals by probe digestion with no help of primers and to amplify signals with no help of simultaneous target amplification reactions may enable to detect multiple target sequences without any problems accounted in the conventional real-time PCR methods such as false positive signals and difficulties in oligonucleotides (primer and probe) selection and reaction condition optimization.
1. A method for detecting a target nucleic acid sequence in a DNA molecule or a mixture of nucleic acids by a cyclic exonucleolytic reaction without the use of an upstream primer, which comprises the steps of:
(a) hybridizing the target nucleic acid sequence with a probe, wherein the probe has a hybridizing nucleotide sequence complementary to a site on the target nucleic acid sequence, a fluorescent reporter molecule, and a quencher molecule capable of quenching the fluorescence of the reporter molecule;
(b) contacting the target-hybridized probe with a template-dependent nucleic acid polymerase having 5′ to 3′ exonuclease activity for cleavage of the probe without the use of an upstream primer, such that either the fluorescent reporter molecule or the quencher molecule is released from the probe to unquench the fluorescence of the reporter molecule and generate a fluorescent signal indicative of the presence of the target nucleic acid sequence; wherein the probe is cleaved by the 5′ to 3′ exonuclease activity of the template-dependent nucleic acid polymerase that is independent from interaction with either an upstream primer or an extended product of an upstream primer;
(c) denaturing a duplex of the probe-target nucleic acid sequence of step (b);
(d) repeating the steps (a)-(c) at least twice to amplify the signal indicative of the presence of the target nucleic acid sequence; and
(e) detecting the signal indicative of the presence of the target nucleic acid sequence, wherein the detection is performed for each cycle of the repetition of step (d), at the end of the repetition of step (d) or at each of predetermined time intervals during the repetition, such that the signal is indicative of the presence of the target nucleic acid sequence.
2. The method according to claim 1 , wherein the polymerase having a 5′ to 3′ exonuclease activity is thermostable.
3. The method according to claim 2 , wherein the method is performed on a solid phase and the probe is immobilized through its 3′-end on the surface of a solid substrate; wherein the probe has a digested portion and an undigested portion by the 5′ to 3′ exonuclease activity of the enzyme such that the undigested portion remains attached to the surface of the solid substrate; wherein the fluorescent reporter molecule on the probe is positioned on the undigested portion and the quencher molecule capable of quenching the fluorescence of the reporter molecule is positioned on the digested portion; wherein when the probe is hybridized with the target nucleic acid sequence, the digested portion of the probe is released from the probe by the 5′ to 3′ exonuclease activity of the polymerase and the fluorescence of the reporter molecule on the undigested portion of the probe is unquenched, whereby a fluorescent signal on the solid substrate is detected to determine the presence of the target nucleic acid sequence.
4. The method according to claim 1 , wherein the probe has a match nucleotide sequence at its 5′-end.
5. The method according to claim 1 , wherein the step (a) is performed using a reverse primer to produce the target nucleic acid sequence hybridizable with the probe in step (b) by an extension reaction of the reverse primer by the template-dependent nucleic acid polymerase.
6. The method according to claim 1 , wherein the target nucleic acid sequence comprises at least two types of nucleic acid sequences and the probe comprises at least two types of probes.