Method and materials for isolation of nucleic acid materials

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.

We claim: 
     
         1 . A method for nucleic acid isolation comprising:
 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, wherein the set of affinity moiety-coated microparticles comprise at least one of PAA and DABAM amide-bonded to a set of microparticles,   contacting the binding moiety solution with a biological sample, within the process chamber, thereby producing a moiety-sample mixture;   incubating the moiety-sample mixture 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;   separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture;   washing the set of moiety-bound nucleic acid particles; and   releasing a nucleic acid sample from the moiety-bound nucleic acid volume with an elution solution characterized by a pH greater than 10.   
     
     
         2 . The method of  claim 1 , wherein receiving the binding moiety solution within the process chamber comprises receiving a set of magnetic microparticles modified with carboxyl functional groups, amide-bonded to at least one of PAA of molecular weight less than 30,000 Da and DABAM (Generation 1) by way of the carboxyl functional groups. 
     
     
         3 . The method of  claim 1 , wherein receiving the binding moiety solution comprises receiving a set of magnetic microparticles covalently bonded to a set of affinity moiety molecules, and wherein contacting the binding moiety solution with the biological sample comprises aspirating the binding moiety solution along with the biological sample from the process chamber and dispensing the binding moiety solution with the biological sample to the process chamber. 
     
     
         4 . The method of  claim 3  wherein incubating the moiety-sample solution comprises heating the moiety-sample solution to a temperature in the range of 25-95 C for 5-60 minutes, thus simultaneously lysing the biological sample and facilitating binding of nucleic acid material of the biological sample to the set of affinity moiety-coated microparticles. 
     
     
         5 . The method of  claim 3 , wherein separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture. 
     
     
         6 . The method of  claim 5 , wherein magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises aspirating the moiety-sample mixture; occluding a fluidic pathway of a microfluidic cartridge at a set of occlusion positions; and delivering the moiety-sample mixture into the fluidic pathway, wherein the fluidic pathway is configured to pass through a magnetic field and is coupled to a waste chamber, such that the set of moiety-bound nucleic acid particles is captured within the magnetic field and a waste volume of the moiety-sample mixture is delivered into the waste chamber. 
     
     
         7 . The method of  claim 1 , wherein separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises at least one of filtering the moiety-sample mixture and focusing the moiety-sample mixture. 
     
     
         8 . The method of  claim 1  wherein receiving the binding moiety solution comprises receiving a binding buffer that is also configured to aid in lysis of nucleic acid-containing cells of the biological sample, thereby releasing nucleic acid material from the biological sample for binding. 
     
     
         9 . The method of  claim 1  wherein receiving the binding moiety solution comprises receiving a binding buffer configured to aid in mitigation of nucleolytic activity from the biological sample. 
     
     
         10 . The method of  claim 3 , wherein washing the set of moiety-bound nucleic acid particles comprises aspirating the moiety-sample mixture; occluding a fluidic pathway of a microfluidic cartridge at a set of occlusion positions; delivering the moiety-sample mixture into the fluidic pathway, wherein the fluidic pathway is configured to pass through a magnetic field and is coupled to a waste chamber, such that the set of moiety-bound nucleic acid particles is captured within the magnetic field; and delivering a wash solution through the fluidic pathway and into the waste chamber in a continuous flow operation. 
     
     
         11 . The method of  claim 1 , wherein washing the set of moiety-bound nucleic acid particles comprises transferring the moiety-sample mixture to a fluid vessel, capturing the set of moiety-bound nucleic acid particles within the fluid vessel, and washing the set of moiety-bound nucleic acid particles using a series of aspiration and dispensing steps. 
     
     
         12 . The method of  claim 11 , wherein washing comprises delivering a wash solution through the fluid vessel at a flow rate between 1 and 10 microliters/second, with a total wash solution volume of between 50 and 2000 microliters. 
     
     
         13 . The method of  claim 3 , wherein releasing the nucleic acid sample from the set of moiety-bound nucleic acid particles comprises occluding a fluidic pathway of a microfluidic cartridge at a set of occlusion positions; delivering the set of moiety-bound nucleic acid particles into the fluidic pathway, wherein the fluidic pathway is configured to pass through a magnetic field; and delivering an elution solution into the fluidic pathway, thereby inducing a pH shift configured to release the nucleic acid sample from the set of moiety-bound nucleic acid particles. 
     
     
         14 . The method of  claim 13 , further comprising capturing the set of moiety-bound nucleic acid particles within the magnetic field and aspirating the nucleic acid sample from the fluidic pathway. 
     
     
         15 . The method of  claim 1 , wherein releasing the nucleic acid sample from the set of moiety-bound nucleic acid particles comprises contacting the set of moiety-bound nucleic acid particles with an elution solution characterized by a pH that is in the range of pH 10 to pH 13. 
     
     
         16 . The method of  claim 13 , wherein releasing the nucleic acid sample comprises eluting the nucleic acid sample into 3-100 uL of the elution solution. 
     
     
         17 . The method of  claim 1 , further comprising processing the nucleic acid sample, wherein processing the nucleic acid sample comprises amplifying nucleic acids of the nucleic acid sample by polymerase chain reaction. 
     
     
         18 . A method for simultaneous isolation and concentration of DNA and RNA from a biological sample, the method comprising:
 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, wherein the set of affinity moiety-coated microparticles comprise at least one of PAA and DABAM amide-bonded to a set of magnetic microparticles;   mixing the binding moiety solution with between 10 uL and 2 mL of a biological sample, within the process chamber, thereby producing a moiety-sample mixture;   incubating the moiety-sample mixture 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;   magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture; and   eluting a nucleic acid sample comprising target DNA and RNA from the set of moiety-bound nucleic acid particles, into less than 20 uL of an elution solution characterized by a pH greater than 10.   
     
     
         19 . The method of  claim 18 , wherein magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises aspirating the moiety-sample mixture; occluding a fluidic pathway of a microfluidic cartridge at a set of occlusion positions; and delivering the moiety-sample mixture into the fluidic pathway, wherein the fluidic pathway is configured to pass through a magnetic field and is coupled to a waste chamber, such that the set of moiety-bound nucleic acid particles is captured within the magnetic field and a waste volume of the moiety-sample mixture is delivered into the waste chamber. 
     
     
         20 . A composition comprising:
 a magnetic microparticle modified with a carboxyl functional group; and   at least one of PAA of molecular weight less than 30,000 Da and DABAM (Generation 1) amide-bonded to the magnetic microparticle by way of the carboxyl functional group.