Enrichment of small nucleic acids

Abstract

Provided herein is technology related to processing samples of nucleic acids and particularly, but not exclusively, to methods for enriching samples for small nucleic acids, such as small circulating cell-free DNA.

We claim: 
     
         1 . A kit, comprising:
 a) a carboxylated magnetic bead;   b) PEG;   c) a silica column; and   d) a wash buffer, comprising:
 i) TWEEN; 
 ii) ethanol; and 
 iii) magnesium chloride. 
   
     
     
         2 . The kit of  claim 1 , wherein said PEG is PEG 8000. 
     
     
         3 . The kit of  claim 2  wherein, said PEG 8000 has a concentration of 4% to 5% weight per volume. 
     
     
         4 . The kit of  claim 2  wherein, said PEG 8000 has a concentration of approximately 4.8%. 
     
     
         5 . The kit of  claim 2  wherein said PEG 8000 has a concentration of approximately 5.1%. 
     
     
         6 . The kit of  claim 1 , wherein a concentration of said PEG is adjusted to provide a desired DNA size selection 
     
     
         7 . The kit of  claim 1 , wherein said TWEEN is TWEEN-20. 
     
     
         8 . The kit of  claim 1 , wherein said wash buffer comprising TWEEN, ethanol and magnesium chloride comprises 10% TWEEN-20, 15% ethanol and 20 mM magnesium chloride. 
     
     
         9 . The kit of  claim 1 , further comprising a wash buffer comprising 70% ethanol. 
     
     
         10 . The kit of  claim 1 , further comprising a solution comprising NaCl. 
     
     
         11 . The kit of  claim 1 , further comprising one or more reagents selected from the group consisting of 5 to 25% ethanol, 5 to 25% methanol, 5 to 25% acetonitrile, 5 to 25% DMSO, 1 to 25% formamide, greater than 1 M NaCl and a chaotropic salt. 
     
     
         12 . The kit of  claim 1 , further comprising one or more reagents or components selected from the group consisting of a reagent to stabilize cells, a reagent to prevent lysis of cells, a cell-stabilizing tube, a preservative, and a material to encapsulate maternal cells. 
     
     
         13 . The kit of  claim 1 , further comprising one or more reagents or components for a) eluting or washing small nucleic acids preferentially from silica; b) retaining large nucleic acids preferentially on silica; c) enriching small nucleic acids by methylated DNA immunoprecipitation with an antibody-coated solid support; d) enriching small nucleic acids by size exclusion; e) enriching small nucleic acids by coefficient of drag alteration sizing; f) enriching small nucleic acids by electrophoresis-based sizing; g) enriching small nucleic acids by solid phase reversible immobilization sizing; h) enriching small nucleic acids by affinity chromatography; or i) enriching small nucleic acids by simultaneous anion exchange and size exclusion. 
     
     
         14 . The kit of  claim 13 , wherein said affinity chromatography comprises microparticles comprising surface irregularities. 
     
     
         15 . The kit of  claim 1 , further comprising one or more reagents for digital PCR, or next-generation sequencing. 
     
     
         16 . A system, comprising:
 a) a carboxylated magnetic bead;   b) PEG;   c) a silica column;   d) a wash buffer, comprising:
 i) TWEEN; 
 ii) ethanol; and 
 iii) magnesium chloride. 
   
     
     
         17 . The system of  claim 15 , further comprising a computer. 
     
     
         18 . The system of  claim 16 , further comprising a programmable robot. 
     
     
         19 . The system of  claim 15 , further comprising a microfluidic apparatus. 
     
     
         20 . The system of  claim 15 , further comprising an apparatus selected from the group consisting of a PCR apparatus, a digital PCR apparatus, a quantitative PCR apparatus, a droplet digital PCR apparatus, a digital counting by sequencing apparatus, a sequencing apparatus, a massively parallel sequencing apparatus, a next-generation sequencing apparatus, a high-throughput shotgun sequencing apparatus, and a mass spectrometry apparatus.