Methods for nucleic acid isolation and kits using solid phase material
First Claim
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1. A method of isolating nucleic acid from a sample, the method comprising:
- providing a sample comprising nucleic acid-containing material and inhibitors;
forming a concentrated region of the sample;
wherein the concentrated region of the sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample;
contacting the separated concentrated region of the sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
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Abstract
The present invention provides methods and kits for isolating nucleic acid from a sample, preferably from a biological sample, using solid phase material.
99 Citations
47 Claims
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1. A method of isolating nucleic acid from a sample, the method comprising:
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providing a sample comprising nucleic acid-containing material and inhibitors;
forming a concentrated region of the sample;
wherein the concentrated region of the sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample;
contacting the separated concentrated region of the sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
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2. A method of isolating nucleic acid from a sample, the method comprising:
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providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
forming a concentrated region of the lysed sample;
wherein the concentrated region of the lysed sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. - View Dependent Claims (40)
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3. A method of isolating nucleic acid from a sample, the method comprising:
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providing a sample comprising cells containing inhibitors and cells containing nuclei;
contacting the biological sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors and form a lysed sample;
forming a concentrated region of the lysed sample;
wherein the concentrated region of the sample comprises nuclei and inhibitors;
substantially separating the concentrated region of the lysed sample from the less concentrated region of the sample;
contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and
separating at least a portion of the nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. - View Dependent Claims (42)
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4. A method of isolating nucleic acid from a sample, the method comprising:
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providing a microfluidic device comprising a loading chamber, a valved process chamber, and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and inhibitors;
placing the sample in the loading chamber;
transferring the sample to a valved process chamber;
forming a concentrated region of the sample in the valved process chamber, wherein the concentrated region of the sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample;
transferring the separated concentrated region of the sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material;
wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
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5. A method of isolating nucleic acid from a sample, the method comprising:
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providing a microfluidic device comprising a loading chamber, a valved process chamber, and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to a valved process chamber;
forming a concentrated region of the lysed sample in the valved process chamber, wherein the concentrated region of the lysed sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material;
wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. - View Dependent Claims (6, 7, 8, 9, 44)
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10. A method of isolating nucleic acid from a sample, the method comprising:
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providing a microfluidic device comprising a loading chamber, a valved process chamber, and a separation chamber comprising a solid phase material;
providing a sample comprising cells containing inhibitors and cells containing nuclei;
placing the sample in the loading chamber;
contacting the sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors to form a lysed sample;
transferring the lysed sample to a valved process chamber;
forming a concentrated region of the lysed sample in the valved process chamber, wherein the concentrated region of the lysed sample comprises nuclei and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material;
wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and
separating at least a portion of the nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 46)
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23. A method of isolating nucleic acid from a sample, the method comprising:
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providing a sample comprising nucleic acid and inhibitors;
contacting the sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing nucleic acid-containing material, if present, to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
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24. A method of isolating nucleic acid from a sample, the method comprising:
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providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant. - View Dependent Claims (41)
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25. A method of isolating nucleic acid from a sample, the method comprising:
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providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and
after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid. - View Dependent Claims (26, 27, 43)
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28. A method of isolating nucleic acid from a sample, the method comprising:
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providing a microfluidic device comprising a loading chamber and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the sample with the solid phase material;
wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and
separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant. - View Dependent Claims (45)
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29. A method of isolating nucleic acid from a sample, the method comprising:
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providing a microfluidic device comprising a loading chamber and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material;
wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and
separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and
after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 47)
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Specification