Purification of DS-DNA using heteropolymeric capture probes and a triplex, quadruplex or homologous duplex binding mechanism
A purification method includes bonding a probe to a target in a sample to form a complex, and separating the sample from the complex to separate in a sequence specific manner the target from the sample. The complex, which is immobilized on a support, is a duplex, triplex or quadruplex formed by Watson-Crick complementary base interaction or by homologous base interaction, provided that when the complex is a duplex and the heteropolymeric probe sequence is antiparallel to the heteropolymeric target sequence, the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by homologous base interaction, and provided that when the complex is a triplex, the complex is free of recombination proteins. A kit for performing the method includes the support and the probe.
1. A method for capturing and separating a target from a sample, said method comprising:
providing the sample comprising the target, wherein the target contains a heteropolymeric target sequence of nucleic acids or nucleic acid analogues;
providing a probe comprising a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues;
bonding the heteropolymeric target sequence to the heteropolymeric probe sequence to provide a complex;
attaching the probe to a support before, during or after the complex is provided; and
separating the sample from the complex to separate the target from the sample,
wherein the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by Watson-Crick complementary base interaction or by homologous base interaction, provided that when the complex is a duplex and the heteropolymeric probe sequence is antiparallel to the heteropolymeric target sequence, the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by homologous base interaction, and provided that when the complex is a triplex, the complex is free of recombination proteins.
2. The method of claim 1 , wherein the probe and the target are single-stranded and the complex is a duplex.
3. The method of claim 1 , wherein one of the probe and the target is single-stranded, the other of the probe and the target is double-stranded, and the complex is a triplex.
4. The method of claim 1 , wherein the probe and the target are double-stranded and the complex is a quadruplex.
5. The method of claim 1 , wherein at least one of the probe and the target is double-stranded and the complex is a triplex or a quadruplex.
6. The method of claim 1 , wherein the complex is immobilized by the support, and the separating of the sample from the complex comprises washing the sample, the immobilized complex and the support with a liquid.
7. The method of claim 6 , wherein the support is a bead, a plate, a membrane or a film.
8. The method of claim 7 , wherein the probe contains biotin, the support is at least partially coated with streptavidin and the complex is attached to the support by linking the biotin to the streptavidin.
9. The method of claim 1 , wherein the sample further comprises at least one member selected from the group consisting of proteins, lipopolysaccharides, nucleases and additional nucleic acid sequences other than the heteropolymeric target sequence.
10. The method of claim 1 , wherein the probe invades the target, displacing a sequence of bases bound to the heteropolymeric target sequence, such that bases of the heteropolymeric probe sequence bind with bases of the heteropolymeric target sequence on the basis of Watson-Crick base recognition or homologous base recognition.
11. The method of claim 1 , wherein the method is conducted without denaturing the probe or the target.
12. The method of claim 1 , wherein the sample is contacted with the target in a liquid medium further comprising at least one binding promoter.
13. The method of claim 12 , wherein the at least one binding promoter is a condensing agent or a decondensing agent.
14. The method of claim 12 , wherein the at least one binding promoter is a member selected from the group consisting of YOYO-1, TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1, cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5, PO-PRO-1, BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1, cyanine monomers, ethidium bromide, ethidium homodimer-1, ethidium homodimer-2, ethidium derivatives, acridine, acridine orange, acridine derivatives, ethidium-acridine heterodimer, ethidium monoazide, propidium iodide, SYTO dyes, SYBR Green 1, SYBR dyes, Pico Green, SYTOX dyes and 7-aminoactinomycin D.
15. The method of claim 1 , further comprising separating the target from the probe after the sample has been separated from the complex.
16. The method of claim 1 , wherein the target is brought into proximity with the probe by moving the sample over or past the support at least once.
17. The method of claim 1 , carried out in solution, on a biochip, in a capillary, in a microwell channel or in a column.
18. The method of claim 1 , further comprising quantitating the target.