Purification Chemistries and Formats for Sanger DNA Sequencing Reactions on a Micro-Fluidics Device

Abstract

According to various embodiments described herein, a microfiuidics-chip based purification device and system for Sanger-sequencing reactions is provided. The device and system allow for the introduction into a sequencing system of a cartridge containing purification technologies specific to the sequencing contaminants or sequencing method where the simplified purification solution of a cartridge allows automation of the sample purification process, reduced consumption of purification reagents, and consistency in sampling by reducing the sampling errors and artifacts. These various embodiments therefore solve the need for a microfiuidics-chip-based, Sanger-sequencing reaction purification system for CE devices. The microfiuidic chips described can be used as a PCR chip by reorganizing the on-chip reagents, reaction wells and work flow steps.

1 . A microfluidic sequencing reaction purification device for reducing the number of sequencing contaminants in a single-stranded DNA sequencing sample, the microfluidics chip-based sequencing reaction purification device comprising,
 a surface;   a solid-phase extraction substrate; and   silane bound to a structure selected from the group consisting of microstructures, the surface of the microfluidic device, a membrane, a high-surface area, convoluted material, and combinations thereof.   
     
     
         2 . The microfluidic sequencing reaction purification device of  claim 1 , wherein the microstructure comprises microbeads, tubes, or plates. 
     
     
         3 . The microfluidic sequencing reaction purification device of  claim 1 , wherein the microstructures comprise polystyrene, latex, agarose, an ion-exchange resin, an immobilized metal affinity chromatography resin, or any other substrate or resin capable of being coated by or bound to silane. 
     
     
         4 . The microfluidic sequencing reaction purification device of  claim 2 , wherein the microstructures are nonmagnetic, magnetic, or paramagnetic. 
     
     
         5 . The microfluidic sequencing reaction purification device of  claim 1 , wherein the high-surface area, convoluted material comprises frit or wool. 
     
     
         6 . The microfluidic sequencing reaction purification device of  claim 1 , wherein the sequencing contaminants comprise salts, free salts, salt ions, ions, sequencing amplification compounds, short primers, dNTPs, enzymes, short-failed Polymerase Chain Reaction/Capillary Electrophoresis products, salts from Polymerase Chain Reaction/Capillary Electrophoresis products, or unincorporated dideoxyNTPs. 
     
     
         7 . The microfluidic based sequencing reaction purification device of  claim 1 , wherein, after the sequencing reaction is completed, the device is configured to introduce unbound silane into the DNA sequencing sample. 
     
     
         8 .- 25 . (canceled) 
     
     
         26 . The microfluidic sequencing reaction purification device of  claim 7 , wherein the reagent comprises a binding buffer. 
     
     
         27 . The microfluidic sequencing reaction purification device of  claim 26 , wherein the high-surface area, convoluted material comprises frit or wool. 
     
     
         28 . The microfluidic sequencing reaction purification device of  claim 26 , wherein the microstructures comprise microbeads, tubes, or plates. 
     
     
         29 . The microfluidic sequencing reaction purification device of  claim 26 , wherein the microstructures comprise polystyrene, latex, agarose, an ion-exchange resin, an immobilized metal affinity chromatography resin, or any other substrate or resin capable of being coated by or bound to the ion exchanger. 
     
     
         30 . The microfluidic sequencing reaction purification device of  claim 26 , wherein the sequencing contaminants comprise salts, free salts, salt ions, ions, sequencing amplification compounds, short primers, dNTPs, enzymes, short-failed Polymerase Chain Reaction/Capillary Electrophoresis products, salts from Polymerase Chain Reaction/Capillary Electrophoresis products, or unincorporated dideoxyNTPs. 
     
     
         31 .- 32 . (canceled) 
     
     
         33 . The microfluidic sequencing reaction purification device of  claim 7 , wherein the reagent comprises a hybridization-based pull-out oligonucleotide selected to be complementary to a sequencing contaminant. 
     
     
         34 . The microfluidic sequencing reaction purification device of  claim 33 , wherein the high-surface area, convoluted material comprises frit or wool. 
     
     
         35 . The microfluidic sequencing reaction purification device of  claim 33 , wherein the microstructures comprise microbeads, tubes, or plates. 
     
     
         36 . The microfluidic sequencing reaction purification device of  claim 33 , wherein the microstructures comprise polystyrene, latex, agarose, an ion-exchange resin, an immobilized metal affinity chromatography resin, or any other substrate or resin capable of being coated by or bound to the ion exchanger. 
     
     
         37 . The microfluidic sequencing reaction purification device of  claim 33 , wherein the sequencing contaminants comprise salts, free salts, salt ions, ions, sequencing amplification compounds, short primers, dNTPs, enzymes, short-failed Polymerase Chain Reaction/Capillary Electrophoresis products, salts from Polymerase Chain Reaction/Capillary Electrophoresis products, or unincorporated dideoxyNTPs. 
     
     
         38 . The microfluidic sequencing reaction purification device of  claim 33 , wherein the microstructures are nonmagnetic, magnetic, or paramagnetic. 
     
     
         39 . The microfluidic sequencing reaction purification device of  claim 33 , wherein, after the sequencing reaction is completed, the device is configured to introduce into the DNA sequencing sample unbound hybridization-based pull-out oligonucleotides selected to be complementary to the sequencing contaminants. 
     
     
         40 . The microfluidic sequencing reaction purification device of  claim 7 , wherein the reagent comprises any combination of silane, an ion exchanger, a reagent adapted to sequester the sequencing contaminants based on the size and charge of the sequencing contaminants, a binding buffer, or a hybridization-based pull-out oligonucleotide selected to be complementary to a sequencing contaminant, as well as their unbound analogues. 
     
     
         41 .- 48 . (canceled)