Triple-stranded nucleic acid comprising tfo, kits and methods for detecting target sequence by using tfo

A method of detecting a target sequence in a double-stranded nucleic acid, comprising

• (a) mixing the double-stranded nucleic acid with polymerase chain reaction (PCR) reagents that comprise a forward primer, a reverse primer, a DNA polymerase, a reaction buffer, and deoxynucleotide triphosphates (dNTPs), wherein
• the forward primer is linked to a first fluorophore;
• the dNTPs consist of a deoxyadenosine triphosphate (dATP), a deoxycytidine triphosphate (dCTP), a deoxyguanosine triphosphate (dGTP), and a modified deoxyuridine triphosphate (dUTP) selected from the group consisting of 5-bromo-dUTP, 5-chloro-dUTP, 5-fluoro-dUTP and 5-formyl-dUTP;
• and
• the PCR reagents do not comprise a deoxythymidine triphosphate (dTTP);
• (b) subjecting the mixture of step (a) to PCR;
• (c) mixing the amplified product of step (b) with a triplex forming oligonucleotide (TFO) thereby forming a triplex complex, wherein the TFO is linked to a second fluorophore, wherein one of the first and second fluorophores is a donor fluorophore, and the other of the first and second fluorophores is an acceptor fluorophore;
• (d) irradiating the product of step (c) with a light having a first wavelength to excite the donor fluorophore;
• and
• (e) measuring the signal emitted from the acceptor fluorophore at a second wavelength to detect the target sequence.

A kit for detecting a target sequence in a double-stranded nucleic acid, comprising

• a forward primer and a reverse primer configured to amplify the target sequence in a polymerase chain reaction (PCR), wherein the forward primer is linked to a first fluorophore;
• a triplex forming oligonucleotide (TFO) having 9-40 nucleotides in length, wherein the nucleotide sequence of the TFO is substantially reverse to a strand amplified by the forward primer, wherein the TFO is linked to a second fluorophore, wherein one of the first and second fluorophores is a donor fluorophore, and the other of the first and second fluorophores is an acceptor fluorophore;
• and
• deoxynucleotide triphosphates (dNTPs) consisting of a deoxyadenosine triphosphate (dATP), a deoxycytidine triphosphate (dCTP), a deoxyguanosine triphosphate (dGTP), and a modified deoxyuridine triphosphate (dUTP) selected from the group consisting of 5-bromo-dUTP, 5-chloro-dUTP, 5-fluoro-dUTP and 5-formyl-dUTP;
• wherein the kit does not comprise a deoxythymidine triphosphate (dTTP).