Nucleic acid capture method
A method and a kit for enriching target nucleic acid sequences from a biological sample are disclosed. The method includes preparing, and contacting with the biological sample, a first RNA probe set and a second RNA probe set respectively and concurrently targeting both of the two antiparallel strands of a duplex segment in each target nucleic acid sequence. Each RNA probe in the first and second RNA probe set can be generated by chemical synthesis or by in vitro or in vivo transcription, and can be biotin-labelled to thereby allow capturing of the target nucleic acid sequences by magnetic beads labelled with streptavidin, or can be engineered to a microfluidic channel to facilitate the capturing. The method can be applied to capture double-stranded nucleic acid sequences or single-stranded nucleic acid sequences having duplex segments, and the nucleic acid sequences can include DNAs, RNAs, or DNA-RNA hybrid molecules.
1 . A method for enriching at least one target nucleic acid sequence each in a polynucleotide from a biological sample, wherein the polynucleotide comprises one or more other sequences, the method comprising:
a) a providing step: providing at least one pair of RNA probe sets and a solid support having a working surface, wherein:
i) the solid support comprises a working surface functionalized with coupling partners to bind immobilization portions of the RNA probes;
ii) each of the at least one pair of RNA probe sets comprises a first RNA probe set and a second RNA probe set configured to concurrently and respectively target two antiparallel strands of a duplex segment in each of the at least one target nucleic acid sequence;
iii) each RNA probe in any of the first RNA probe set and the second RNA probe set comprises an immobilization portion for attachment to the solid support; and
b) a capturing step: capturing the two antiparallel strands of the duplex segment from the biological sample, wherein:
i) the two antiparallel strands of the duplex segment in the at least one target nucleic acid sequence are hybridized concurrently and respectively to the first RNA probe set and the second RNA probe set in the at least one pair of RNA probe sets; and
ii) the immobilization portion of each RNA probe binds to the coupling partners on the solid support during or after hybridization, thereby capturing the target nucleic acid sequence on the solid support;
c) a blocking step: blocking non-target sequences, wherein prior to or concurrent with the hybridization:
i) at least one blocking oligonucleotide is hybridized with the polynucleotide in the biological sample, such that the at least one blocking oligonucleotide hybridizes with and blocks one strand in at least one of the one or more other sequences in the polynucleotide; and
ii) the hybridization of said at least one blocking oligonucleotide sterically or competitively inhibits hybridization of RNA probes to non-target sequences and thereby reduces non-specific binding of the RNA probes.
2 . The method of claim 1 , wherein each RNA probe in any of the first RNA probe set and the second RNA probe set in the each of the at least one pair of RNA probe sets has a length of about 100-150 nucleotides (nt), wherein:
the contacting both the first RNA probe set and the second RNA probe set in the each of the at least one pair of RNA probe sets with the at least one target nucleic acid sequence in the biological sample is performed at a temperature of about 62-70° C.
3 . The method of claim 1 , wherein the first RNA probe set and the second RNA probe set in the each of the at least one pair of RNA probe sets respectively target a different portion of the duplex segment in the each of the at least one target nucleic acid sequence.
4 . The method of claim 1 , wherein the first RNA probe set and the second RNA probe set in each pair respectively hybridize to complementary regions on the two antiparallel strands of the same region of the at least one target nucleic acid sequence.
5 . The method of claim 4 , wherein the hybridization occurs in a single hybridization reaction, wherein contacting both the first RNA probe set and the second RNA probe set are concurrently hybridized to the two antiparallel strands of the duplex segment in each of the at least one target nucleic acid sequence.
6 . The method of claim 5 , wherein, for the at least one pair of RNA probe sets, each probe in the first RNA probe set and each probe in the second RNA probe set are immobilized at physically separated positions on the working surface of the solid support.
7 . The method of claim 5 , wherein in the each of the at least one pair of RNA probe sets, each probe in the first RNA probe set and each probe the second RNA probe set are not physically separated from one another.
8 . The method of claim 4 , wherein the contacting both the first RNA probe set and the second RNA probe set in the each of the at least one pair of RNA probe sets with the at least one target nucleic acid sequence in the biological sample comprises sequentially:
contacting one of the first RNA probe set and the second RNA probe set in each of the at least one pair of RNA probe sets with the at least one target nucleic acid sequence in a first hybridization reaction; and
contacting another of the first RNA probe set and the second RNA probe set in each of the at least one pair of RNA probe sets with the at least one target nucleic acid sequence in a second hybridization reaction.
9 . The method of claim 4 , wherein the contacting both the first RNA probe set and the second RNA probe set in the each of the at least one pair of RNA probe sets with the at least one target nucleic acid sequence in the biological sample comprises at least one round of:
separately contacting the first RNA probe set and the second RNA probe set in each of the at least one pair of RNA probe sets with the at least one target nucleic acid sequence in a third hybridization reaction and a fourth hybridization reaction, respectively; and
combining the third hybridization reaction and the fourth hybridization reaction to thereby allow a fifth hybridization reaction to proceed.
10 . The method of claim 1 , wherein the RNA probes in each of the at least one pair of RNA probe sets are attached to immobilized on the working surface of the solid support prior to the capturing step to form at least one pair of immobilized RNA probe sets, each pair comprising an immobilized first RNA probe set and an immobilized second RNA probe set; and wherein in the capturing step, the immobilized first RNA probe set and the immobilized second RNA probe set hybridize to the two antiparallel strands of the duplex segment in each of the at least one target nucleic acid sequence.
11 . The method of claim 10 , wherein the working surface is selected from the group consisting of a magnetic bead, a filter, a resin bead, a nanosphere, a plastic surface, a microtiter plate, a glass surface, a slide, a membrane, a microfluidic channel, a chip, and a matrix, wherein the solid support can be labelled, conjugated, or attached with a coupling partner corresponding to the immobilization portion.
12 . The method of claim 10 , wherein the immobilized first RNA probe set and the immobilized second RNA probe set:
Concurrently hybridize to the two antiparallel strands of each of the at least one target nucleic acid sequence.
13 . The method of claim 10 , wherein the contacting both the immobilized first RNA probe set and the immobilized second RNA probe set in the each of the at least one pair of immobilized RNA probe sets with the at least one target nucleic acid sequence comprises sequentially:
contacting one of the immobilized first RNA probe set and the immobilized second RNA probe set in the each of the at least one pair of immobilized RNA probe sets with the at least one target nucleic acid sequence in a sixth hybridization reaction; and
contacting another of the immobilized first RNA probe set and the immobilized second RNA probe set in the each of the at least one pair of immobilized RNA probe sets with the at least one target nucleic acid sequence in a seventh hybridization reaction.
14 . The method of claim 10 , wherein the contacting both the immobilized first RNA probe set and the immobilized second RNA probe set in the each of the at least one pair of immobilized RNA probe sets with the at least one target nucleic acid sequence comprises:
separately contacting the immobilized first RNA probe set and the immobilized second RNA probe set in the each of the at least one pair of immobilized RNA probe sets with the at least one target nucleic acid sequence in an eighth hybridization reaction and a ninth hybridization reaction, respectively; and
combining the eighth hybridization reaction and the ninth hybridization reaction to thereby allow a tenth hybridization reaction to proceed.