Apparatus and method for specific release of captured extension products
First Claim
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1. An apparatus for separating one or more different-sequence polynucleotides from a polynucleotide mixture, comprising:
- (a) a flow path, (b) a plurality of solid supports which are disposed in series in the flow path, each support having bound thereto a sequence-specific capture agent that is complementary to a different-sequence target that may be present in the polynucleotide mixture, and (c) a control mechanism in communication with the supports for altering a physical property of each support, separately from the other supports, between a target-binding state and a target-nonbinding state, whereby (i) passage of such a mixture through the plurality of solid supports is effective to specifically bind different-sequence targets to a complementary capture agent on each support when the supports are each in a target-binding state, (ii) alteration of a physical property of a first selected support to a target-non-binding state is effective to release bound polynucleotides from that support, (iii) the polynucleotides released thereby can be eluted from that support by passage of a liquid medium through the flow path while polynucleotides captured on the other supports remain bound to those supports, and (iv) bound polynucleotides on the remaining supports can be released and eluted separately by repetition of steps (ii) and (iii) on the remaining supports.
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Abstract
Apparatus and methods for separating different polynucleotide populations in a mixture are provided, wherein different polynucleotides or polynucleotide populations are captured on different solid support. After hybridization, polynucleotides are selectively released from a selected support by altering a physical property of that support. The released polynucleotides can be eluted from a common flow path and isolated.
16 Citations
20 Claims
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1. An apparatus for separating one or more different-sequence polynucleotides from a polynucleotide mixture, comprising:
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(a) a flow path, (b) a plurality of solid supports which are disposed in series in the flow path, each support having bound thereto a sequence-specific capture agent that is complementary to a different-sequence target that may be present in the polynucleotide mixture, and (c) a control mechanism in communication with the supports for altering a physical property of each support, separately from the other supports, between a target-binding state and a target-nonbinding state, whereby (i) passage of such a mixture through the plurality of solid supports is effective to specifically bind different-sequence targets to a complementary capture agent on each support when the supports are each in a target-binding state, (ii) alteration of a physical property of a first selected support to a target-non-binding state is effective to release bound polynucleotides from that support, (iii) the polynucleotides released thereby can be eluted from that support by passage of a liquid medium through the flow path while polynucleotides captured on the other supports remain bound to those supports, and (iv) bound polynucleotides on the remaining supports can be released and eluted separately by repetition of steps (ii) and (iii) on the remaining supports. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for isolating one or more different-sequence polynucleotides from a mixture, the method comprising:
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(a) flowing the mixture through a flow path containing a plurality of solid supports which are located in series in the flow path, each support having bound thereto a sequence-specific capture agent complementary to a different-sequence polynucleotide, under conditions effective to specifically bind different-sequence polynucleotides to corresponding sequence-specific capture agents on one or more of the supports, (b) after said specific binding, releasing bound polynucleotides from a selected support by altering a physical property of that support while leaving unaltered the same physical property of at least one other of the supports, (c) eluting the released polynucleotides through the flow path such that the eluted polynucleotides can be isolated in separated form. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification