Method and materials for isolation of nucleic acid materials

Inventors

Brahmasandra, Sundaresh • Mastronardi, Michelle • Craig, Elizabeth • Carey, Maureen

Assignees

NeumoDx Molecular Inc

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 nucleic acid isolation method in which a binding moiety solution is received within a process chamber. The binding moiety solution comprises a collection buffer and a set of affinity moiety-coated microparticles, where the affinity moiety-coated microparticles comprise Polypropylenimine tetramine dendrimer Generation 1 amide-bonded to a set of microparticles. A moiety-sample mixture is produced by contacting the binding moiety solution with a biological sample within the process chamber.

The moiety-sample mixture is incubated to reversibly bind nucleic acid material of the biological sample to the set of affinity moiety-coated microparticles, thereby producing a set of moiety-bound nucleic acid particles. The workflow uses a cartridge receiving module that includes a set of pins, each pin displaceable between a first position and a second position. Upon displacement of a first subset of the set of pins, the method occludes a fluidic pathway of the cartridge to separate the moiety-bound nucleic acid particles from the moiety-sample mixture within the fluidic pathway.

The invention further includes washing and releasing using coordinated pin displacement to modulate flow through the cartridge fluidic pathway. Washing occurs within the fluidic pathway of the cartridge in coordination with displacement of a second subset of the set of pins, where 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. Releasing a nucleic acid sample from the moiety-bound nucleic acid particles is performed with an elution solution characterized by a pH greater than 10, including in coordination with displacement of the second subset of the pins.

In one aspect, the method provides total nucleic acid extraction by simultaneously isolating and concentrating DNA and RNA, including binding target DNA and RNA to the affinity moiety-coated microparticles and eluting the nucleic acid sample into less than 20uL of an elution solution characterized by a pH greater than 10. In another aspect, the affinity moiety-coated microparticles include set of microparticles amide-bonded to at least one of Poly(allylamine) of molecular weight <30,000 Da and Polypropylenimine tetramine dendrimer Generation 1. In certain mechanical implementations, occlusion of the fluidic pathway is achieved by linearly displacing a cam card relative to the pins such that at least one pin transitions between the first and second positions.

Claims Coverage

The provided material includes three independent claims. Across the independent claims, there are core inventive features covering a specific affinity moiety-coated microparticle binding system, reversible nucleic acid binding in a process chamber, and a cartridge receiving module with displaceable pins to occlude fluidic pathways for separating, washing, and eluting nucleic acids, using elution at pH greater than 10.

Across the independent claims, the coverage centers on reversible nucleic acid binding using specified amide-bonded affinity moiety-coated microparticles, combined with cartridge fluidic control via displaceable pins to occlude fluidic pathways for separating, washing, and eluting. Elution is characterized by pH greater than 10, and in one independent claim total nucleic acid extraction includes simultaneous DNA and RNA isolation and concentration with elution into less than 20uL.

Stated Advantages

Documented Applications