TSG primer target detection

A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids using a target signal generating primer (TSG primer) in an amplification reaction:

• wherein said target signal generating primer (TSG primer) is not immobilized on a solid substrate;
• and
• wherein said amplification reaction is in a liquid phase;
• said method comprising the steps:
• (a) hybridizing the target nucleic acid sequence with a primer pair composed of two primers as a forward primer and a reverse primer capable of amplifying the target nucleic acid sequence;
• wherein at least one of the two primers is the TSG primer;
• wherein the TSG primer comprises (i) a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a dual label system consisting of a single reporter molecule and a single non-fluorescent quencher molecule;
• wherein either or both of the reporter molecule and the quencher molecule is/are located on the hybridizing nucleotide sequence;
• wherein the reporter molecule and the quencher molecule are positioned at 6-40 nucleotides apart from each other;
• wherein the TSG primer is a single stranded oligonucleotide and has no substantial self-complementary sequence such that the TSG primer does not comprise hairpin loop structure(s) when it is not hybridized with the target nucleic acid sequence;
• wherein when the TSG primer is not hybridized with the target nucleic acid sequence, the reporter molecule and the quencher molecule are three-dimensionally adjacent to each other in a twist conformation, without assist by hairpin loop structure(s), to allow the quencher molecule to quench a signal from the reporter molecule, thereby generating no signal indicative of the presence of the target nucleic acid sequence;
• wherein when the TSG primer is hybridized with the target nucleic acid sequence, the reporter molecule and the quencher molecule are three-dimensionally separated in a stretch conformation to allow the quencher molecule to unquench the signal from the reporter molecule, whereby the signal indicative of the presence of the target nucleic acid sequence is generated and obtained without cleavage of the TSG primer before extension of the primer pair wherein the TSG primer does not comprise a minor groove binder (MGB);
• (b) contacting the resultant of step (a) to a template-dependent nucleic acid polymerase under primer extension conditions such that the 3′-extension reaction at the 3′-ends of the two primers is induced;
• (c) denaturing the resultant of step (b);
• (d) repeating the steps (a)-(c) at least twice to amplify both the target nucleic acid sequence and 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 indicates the presence of the target nucleic acid sequence;
• wherein at least some of the amplified target nucleic acid products comprises the reporter molecule and the quencher molecule remaining attached to the TSG primer, and the reporter molecule and the quencher molecule on the amplified target nucleic acid products provide the signal indicative of the presence of the target nucleic acid sequence;
• and wherein the method does not further include any other oligonucleotide comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence.