Method and materials for isolation of nucleic acid materials
Inventors
Brahmasandra, Sundaresh • Mastronardi, Michelle • Craig, Elizabeth • Carey, Maureen
Assignees
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Publication Number
US-9540636-B2
Publication Date
2017-01-10
Expiration Date
Abstract
A method for nucleic acid isolation comprising: receiving a binding moiety solution within a process chamber; mixing the binding moiety solution with a biological sample, within the process chamber, in order to produce a moiety-sample mixture; incubating the moiety-sample mixture during a time window, thereby producing a solution comprising a set of moiety-bound nucleic acid particles and a waste volume; separating the set of moiety-bound nucleic acid particles from the waste volume; washing the set of moiety-bound nucleic acid particles; and releasing a nucleic acid sample from the set of moiety-bound nucleic acid particles. The method preferably utilizes a binding moiety comprising at least one of poly(allylamine) and polypropylenimine tetramine dendrimer, both of which reversibly bind and unbind to nucleic acids based upon environmental pH.
Core Innovation
The invention provides a method for nucleic acid isolation that introduces a binding moiety solution into a process chamber, where the binding moiety solution includes a collection buffer and a set of affinity moiety-coated microparticles. The binding moiety solution is contacted with a biological sample in the process chamber to form a moiety-sample mixture, and the moiety-sample mixture is incubated so that nucleic acid material reversibly binds to the set of affinity moiety-coated microparticles, producing a set of moiety-bound nucleic acid particles.
A cartridge is received at a cartridge receiving module that includes a set of pins displaceable between first and second positions. Displacement of a first subset of the pins occludes a fluidic pathway within the cartridge, and the moiety-bound nucleic acid particles are separated from the moiety-sample mixture within the fluidic pathway and then washed within the fluidic pathway while displacement of a second subset of pins coordinates with reversing occlusion of at least a portion of the fluidic pathway.
In related embodiments, the nucleic acid material includes target DNA and target RNA, and separation is performed by magnetically separating the moiety-bound nucleic acid particles from the moiety-sample mixture within the cartridge fluidic pathway. Releasing nucleic acids is performed using an elution solution, and washing and release are integrated with cartridge fluid handling controlled by pin displacement. The described polymer-and-dendrimer approach emphasizes reversible binding and unbinding controlled by environmental pH, using cationic polyelectrolytes including poly(allylamine) (PAA) and polypropylenimine tetramine dendrimer (DABAM) with constraints intended to avoid irreversible binding.
Claims Coverage
The partial content identifies three independent claims. Across them, the claims cover reversible binding to affinity moiety-coated microparticles in a process chamber, cartridge-based fluidic pathway control using displaceable pins that occlude and de-occlude the pathway to coordinate separation and washing, and embodiments including magnetic separation, target DNA and target RNA capture, and release using an elution solution.
Reversible binding using affinity moiety-coated microparticles in a process chamber
Receiving a binding moiety solution within a process chamber, wherein the binding moiety solution comprises a collection buffer and a set of affinity moiety-coated microparticles, contacting the binding moiety solution with a biological sample to produce a moiety-sample mixture, and incubating the moiety-sample mixture to reversibly bind nucleic acid material of the biological sample to the set of affinity moiety-coated microparticles to produce a set of moiety-bound nucleic acid particles.
Pin-actuated occlusion of a cartridge fluidic pathway for separation and washing
Receiving a cartridge at a cartridge receiving module comprising a set of pins displaceable between a first position and a second position, occluding a fluidic pathway of the cartridge upon displacement of a first subset of the set of pins, separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture within the fluidic pathway of the cartridge, and washing the set of moiety-bound nucleic acid particles within the fluidic pathway in coordination with displacement of a second subset of pins, wherein displacement of the second subset includes reversal of displacement of at least one pin in the first subset to reverse occlusion of a portion of the fluidic pathway.
Magnetic separation within a pin-occluded cartridge pathway
Receiving a cartridge at a cartridge receiving module comprising a set of pins displaceable between a first position and a second position, occluding a fluidic pathway of the cartridge upon displacement of a first subset of the set of pins, and magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture within the fluidic pathway of the cartridge.
Target DNA and target RNA reversible binding to affinity moiety-coated microparticles
Incubating the moiety-sample mixture to reversibly bind nucleic acid material comprising target DNA and target RNA of the biological sample to the set of affinity moiety-coated microparticles to produce a set of moiety-bound nucleic acid particles.
Release of nucleic acid using an elution solution
Releasing a nucleic acid sample from the set of moiety-bound nucleic acid particles with an elution solution.
The independent claim set centers on reversible nucleic acid binding to affinity moiety-coated microparticles in a process chamber, followed by cartridge-based separation and washing using displaceable pins that occlude and de-occlude a cartridge fluidic pathway. Additional coverage includes magnetic separation, reversible binding of target DNA and target RNA, and nucleic acid release using an elution solution.
Stated Advantages
Enables reversible binding and unbinding of nucleic acid material controlled by environmental pH.
Coordinates separation and washing operations within a cartridge fluidic pathway using pin displacement and reversible occlusion.
Supports magnetic separation of moiety-bound nucleic acid particles within the cartridge fluidic pathway.
Supports releasing nucleic acids using an elution solution.
Documented Applications
Nucleic acid isolation from biological sample for diagnostic processing using polymerase chain reaction (PCR) in a diagnostic chamber.
RT-PCR and real-time PCR performance using sample matrices including CSF, M4/UTM nasal swabs, urine, plasma, buccal swab, and whole blood with targets including Enterovirus RNA and Group B Streptococcus (GBS) DNA.
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