Detection of nucleic acid sequences by cleavage and separation of tag-containing structures
The present invention is directed to a method of detecting pluralities of target nucleic acid sequences by forming and cleaving duplex structures with a pair of probes, one probe of each pair being labeled with an electrophoretic tag. Cleavage of the duplex structures releases electrophoretic tags that are then separated and identified to indicate the presence or quantity of the target sequences. The present invention is particularly useful in multiplex reactions wherein multiple target sequences are detected in one reaction. Kits useful in the detection of nucleic acids are also provided.
1 . A method of detecting a plurality of polynucleotides in a sample, the method comprising the steps of:
providing for each polynucleotide a helper probe complementary to a region of the polynucleotide and an electrophoretic probe complementary to the helper probe and to the polynucleotide adjacent to said region, such that the helper probe and the electrophoretic probe form a recognition duplex upon hybridization to each other and to the polynucleotide, each electrophoretic probe having attached an electrophoretic tag with a separation or detection characteristic distinct from those of other electrophoretic tags so that each electrophoretic tag forms a distinguishable peak in a separation profile;
combining under hybridization conditions the sample, the helper probes, and the electrophoretic probes to form an assay mixture such that recognition duplexes are formed;
cleaving the recognition duplexes at a cleavage site so that electrophoretic tags are released; and
separating and identifying the released electrophoretic tags to detect each of the plurality of polynucleotides.
2 . The method of claim 1 wherein each of said released electrophoretic tags has a molecular weight of from 150 to 10,000 daltons.
3 . The method of claim 2 including, prior to said step of separating, a further step of treating said assay mixture to exclude from said separation profile uncleaved electrophoretic probes.
4 . The method of claim 3 wherein each of said electrophoretic probes has a capture ligand attached to a nucleotide located opposite said cleavage site from said electrophoretic tag and wherein said step of treating further includes reacting the capture ligand with a capture agent.
5 . The method of claim 3 wherein said step of cleaving produces a released electrophoretic tag having a charge opposite that of said uncleaved electrophoretic probe.
6 . The method of claim 3 wherein each of said electrophoretic probes has a quencher attached to a nucleotide located opposite said cleavage site from said electrophoretic tag such that upon separating uncleaved electrophoretic probes generate no signal in said separation profile.
7 . The method of claim 1 , wherein said separation characteristic is electrophoretic mobility and wherein said plurality is in the range from 5 to 100.
8 . The method of claim 7 wherein each of said released electrophoretic tags has a distinct charge/mass ratio in the range of from −0.001 to 0.5.
9 . The method of claim 7 wherein at least one of said released electrophoretic tags has a positive charge.
10 . The method of claim 7 wherein every said released electrophoretic tag has a negative charge.
11 . The method of claim 7 wherein said step of cleaving is carried out by a hOGG 1 protein and said electrophoretic probe is defined by the formula:
3′-(N) j -Z-(B) k -(M,D)
wherein B and N are each a nucleotide; j is an integer in the range of from 8 to 40; k is an integer in the range of from 1 to 3; Z is selected from the group consisting of 7,8-dihydro-8-oxo-2′-deoxyguanosine, foramidopyrimidine guanosine, and methylforamidopyrimidine guanosine; D is a fluorescent dye; and M is a mobility modifying moiety that is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.
12 . The method of claim 11 wherein Z is 7,8-dihydro-8-oxo-2′-deoxyguanosine and wherein at least one N has said capture ligand attached.
13 . A kit for detecting the presence or absence of one or more target polynucleotides in a sample, the kit comprising:
a plurality of pairs of helper probes and electrophoretic probes, each helper probe of a pair being complementary to a region of a target polynucleotide and each electrophoretic probe of the same pair being complementary to the helper probe and to the target polynucleotide adjacent to said region, such that the helper probe and the electrophoretic probe form a recognition duplex upon hybridization to each other and to the target polynucleotide, each electrophoretic probe having attached an electrophoretic tag with a separation or detection characteristic distinct from those of other electrophoretic tags so that each electrophoretic tag forms a distinguishable peak in a separation profile.
14 . The kit of claim 13 further including a cleavage agent for recognizing and cleaving said recognition duplex.
15 . The kit of claim 13 wherein said electrophoretic probe is selected from a group defined by the formula:
(D,M)-T
wherein D is a fluorescent dye; M is a mobility modifying moiety that is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur; and T is an oligonucleotide having a capture ligand attached.
16 . The kit of claim 15 wherein said electrophoretic probe is selected from the group defined by the formula:
3′-(N) j -Z-(B) k -(M,D)
wherein B and N are each a nucleotide; j is an integer in the range of from 8 to 40; k is an integer in the range of from 1 to 3; and Z is selected from the group consisting of 7,8-dihydro-8-oxo-2′-deoxyguanosine, foramidopyrimidine guanosine, and methylforamidopyrimidine guanosine.
17 . The kit of claim 13 , wherein said plurality of pairs of probes is in the range of from 5 to 50.