Single molecule analysis target-mediated ligation of bipartite primers
First Claim
1. A method of amplifying nucleic acid sequences, the method comprising,(a) mixing a half probe with a target sample comprising a target sequence, to produce a probe-target mixture, and incubating the probe-target mixture under conditions that promote hybridization between the half probe and the target sequence in the probe-target mixture, (b) mixing a probe/primer with the probe-target mixture, and incubating the probe-target mixture under conditions that promote hybridization between the probe/primer and the target sequence, (c) mixing ligase with the probe-target mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the half probe and the probe/primer to form a rolling circle replication primer, (d) mixing an amplification target circle with the rolling circle replication primer, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circle and the rolling circle replication primer in the primer-ATC mixture, and (e) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circle, wherein replication of the amplification target circle results in the formation of tandem sequence DNA.
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Accused Products
Abstract
Disclosed are compositions and a method for detecting single nucleic acid molecules using rolling circle amplification (RCA) of single-stranded circular templates, referred to as amplification target circles, primed by immobilized primers. In one form of the method, referred to as a bipartite primer rolling circle amplification, (BP-RCA), RCA of the amplification target circle (ATC) depends on the formation of a primer by target-mediated ligation. In the presence of a nucleic acid molecule having the target sequence, a probe and a combination probe/primer oligonucleotide can hybridize to adjacent sites on the target sequence allowing the probes to be ligated together. By attaching the first probe to a substrate such as a bead or glass slide, unligated probe/primer can be removed after ligation. The only primers remaining will be primers ligated, via the probe portion of the probe/primer, to the first probe. The ligated primer can then be used to prime replication of its cognate ATC. In this way, an ATC will only be replicated if the target sequence (to which its cognate probe/primer is complementary) is present. BP-RCA is useful, for example, for determining which target sequences are present in a nucleic acid sample, or for determining which samples contain a target sequence. In another form of the method, referred to as immobilized primer rolling circle amplification (IP-RCA), RCA of the ATC depends on incorporation of a target sequence in the ATC during its formation. If the target sequence has been incorporated, a primer that can hybridize to the sequence will prime RCA of the ATC. This form of the method is useful for determining which form or forms of a variable sequence are present in a nucleic acid sample.
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Citations
27 Claims
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1. A method of amplifying nucleic acid sequences, the method comprising,
(a) mixing a half probe with a target sample comprising a target sequence, to produce a probe-target mixture, and incubating the probe-target mixture under conditions that promote hybridization between the half probe and the target sequence in the probe-target mixture, (b) mixing a probe/primer with the probe-target mixture, and incubating the probe-target mixture under conditions that promote hybridization between the probe/primer and the target sequence, (c) mixing ligase with the probe-target mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the half probe and the probe/primer to form a rolling circle replication primer, (d) mixing an amplification target circle with the rolling circle replication primer, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circle and the rolling circle replication primer in the primer-ATC mixture, and (e) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circle, wherein replication of the amplification target circle results in the formation of tandem sequence DNA.
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2. The method of claim 1 wherein the half probe is immobilized on a solid-state support.
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3. The method of claim 1 wherein the probe/primer has two free 3′
- ends and no free 5′
end.
- ends and no free 5′
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4. The method of claim 1 wherein the primer portion and the target probe portion of the probe/primer are coupled 5′
- end to 5′
end.
- end to 5′
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5. The method of claim 1 wherein the nucleotides of the primer portion of the probe/primer are all in the 3′
- to 5′
orientation and the nucleotides of the target probe portion of the probe/primer are all in the 5′
to 3′
orientation.
- to 5′
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6. The method of claim 1 wherein the half probe and the probe/primer hybridize to adjacent regions in the target sequence.
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7. The method of claim 1 wherein the half probe and the probe/primer hybridize to different regions in the target sequence such that there is a gap space between the half probe and the probe/primer when they are hybridized to the target sequence,
wherein, prior to ligation, the gap space is filled by hybridization of one or more gap oligonucleotides, by gap-filling synthesis, or by a combination of one or more gap oligonucleotides and gap-filling synthesis.
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8. The method of claim 1 further comprising detecting the tandem sequence DNA, wherein detection of the tandem sequence DNA indicates the presence of the target sequence in the target sample.
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9. The method of claim 1 wherein the half probe comprises a target probe portion,
wherein the probe/primer comprises a primer portion and a target probe portion, wherein the target sequence comprises a 5′ - region and a 3′
region, wherein the 5′
region and the 3′
region are adjacent in the target sequence, andwherein the target probe portion of the half probe and the target probe portion of the probe/primer are complimentary to the 5′
region and the 3′
region, respectively, of the target sequence.
- region and a 3′
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10. The method of claim 1 wherein a plurality of different half probes are mixed with the target sample,
wherein a plurality of different probe/primers are mixed with the probe-target mixture, wherein the target sample contains a plurality of target sequences, wherein each half probe and each probe/primer hybridize to adjacent regions in at least one of the target sequences, wherein a plurality of the half probes and a plurality of the probe/primers are ligated to form a plurality of different rolling circle replication primers, wherein the hybridized amplification target circles are replicated to form a plurality of tandem sequence DNA molecules.
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11. The method of claim 10 further comprising detecting the tandem sequence DNA molecules, wherein detection of the tandem sequence DNA molecules indicates the presence of the target sequences in the target sample.
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12. The method of claim 10 further comprising detecting the tandem sequence DNA molecules, wherein detection of the tandem sequence DNA molecules indicates the presence of the corresponding target sequences in the target sample.
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13. The method of claim 10 wherein the plurality of probe/primers comprise four sets of probe/primers,
wherein each probe/primer comprises a primer portion and a target probe portion, wherein each probe/primer has the structure
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14. The method of claim 13 wherein the nucleotides of the primer portion of the probe/primer are all in the 3′
- to 5′
orientation and the nucleotides of the target probe portion of the probe/primer are all in the 5′
to 3′
orientation.
- to 5′
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15. The method of claim 1 wherein a plurality of different half probes are mixed with the target sample, wherein the half probe comprises a target probe portion,
wherein a plurality of different probe/primers are mixed with the probe-target mixture, wherein the probe/primer comprises a primer portion and a target probe portion, wherein the target sample contains a plurality of target sequences, wherein each target sequence comprises a 5′ - region and a 3′
region, wherein the 5′
region and the 3′
region are adjacent in each target sequence,wherein the target probe portion of each half probe and the target probe portion of each probe/primer are complimentary to the 5′
region and the 3′
region, respectively, of at least one of the target sequences,wherein a plurality of the half probes and a plurality of the probe/primers are ligated to form a plurality of different rolling circle replication primers, wherein the hybridized amplification target circles are replicated to form a plurality of tandem sequence DNA molecules.
- region and a 3′
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16. The method of claim 15 further comprising detecting the tandem sequence DNA molecules, wherein detection of the tandem sequence DNA molecules indicates the presence of the corresponding target sequences in the target sample.
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17. A method of amplifying nucleic acid sequences, the method comprising,
(a) mixing a probe/primer with a target sample comprising a target sequence, to produce a probe-target mixture, and incubating the probe-target mixture under conditions that promote hybridization between the probe/primer and the target sequence, wherein the target sequence is immobilized on a solid-state support, wherein the probe/primer comprises a primer portion and a target probe portion, wherein the probe/primer has two free 3′ - ends and no free 5′
end,(b) mixing a polymerase with the probe-target mixture, to produce an extension mixture, and incubating the extension mixture under conditions that promote extension of the probe/primer from the target probe portion of the probe/primer, (c) mixing an amplification target circle with the probe/primer, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circle and the rolling circle replication primer in the primer-ATC mixture, and (d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circle, wherein replication of the amplification target circle results in the formation of tandem sequence DNA, wherein the target sequence DNA is immobilized on the solid-state support via hybridization between the extended target probe portion of the probe/primer and the target sequence.
- ends and no free 5′
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18. The method of claim 17 wherein the target sample is immobilized through cytological or histological preparation.
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19. The method of claim 17 wherein the 3′
- terminal nucleotide in the target probe portion of the probe/primer corresponds to a nucleotide position in the target sequence that varies in different forms of the target sequence.
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20. A kit comprising four sets of probe/primers,
wherein each probe/primer comprises a primer portion and a target probe portion, wherein each probe/primer has the structure
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21. The kit of claim 20 wherein the nucleotides of the primer portion of the probe/primer are all in the 3′
- to 5′
orientation and the nucleotides of the target probe portion of the probelprimer are all in the 5′
to 3′
orientation.
- to 5′
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22. The kit of claim 20 further comprising a plurality of amplification target circles,
wherein each amplification target circle comprises a primer complement portion, wherein the primer complement portion of each amplification target circle is complementary to the primer portion of one or more of the probe/primers.
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23. The kit of claim 22 further comprising a plurality of collapsing detection probes each comprising a sequence matching a sequence in one or more of the amplification target circles.
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24. The kit of claim 23 wherein all of the probe/primers in each set of probe/primers has the same primer portion, wherein the primer portion is different for each set of probe/primers,
wherein there are four amplification target circles, each with a different primer complement portion complementary to one of the primer portions, wherein there are four collapsing detection probes, each comprising a different sequence matching a sequence in one of the amplification target circles.
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25. A method of amplifying nucleic acid sequences, the method comprising,
(a) mixing one or more different open circle probes with a target sample comprising one or more target sequences, to produce an OCP-target sample mixture, wherein the target sequences each comprise a 5′ - region and a 3′
region,wherein the open circle probes each comprise a single-stranded, linear DNA molecule comprising, from 5′
end to 3′
end, a 5′
phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′
hydroxyl group, wherein the spacer portion comprises a primer complement portion, and wherein the left target probe portion and the right target probe portion of the same open circle probe are each complementary to the 3′
region and the 5′
region, respectively, of the same target sequence,wherein at least one of the target sequences further comprises a central region located between the 5′
region and the 3′
region,wherein neither the left target probe portion of the open circle probe nor the right target probe portion of any of the open circle probes is complementary to the central region of the target sequences, and incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the target sequences in the OCP-target sample mixture, (b) mixing ligase and DNA polymerase with the OCP-target sample mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the open circle probes to form amplification target circles, wherein during incubation the DNA polymerase fills in the central region of the target sequences, (c) mixing one or more rolling circle replication primers with the ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture, wherein the one or more rolling circle replication primers are immobilized on a solid-state support, and (d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circle results in the formation of tandem sequence DNA.
- region and a 3′
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26. The method of claim 25 wherein sequences in the one or more rolling circle replication primers match the central region of one or more of the target sequences.
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27. The method of claim 20 wherein the probe/primers each have two free 3′
- ends and no free 5′
end.
- ends and no free 5′
Specification