Systems and methods for isolating nucleic acids

Abstract

The present disclosure relates to systems and methods for nucleic acid isolation. In particular, the present disclosure provides systems and methods for isolating low molecular weight circulating nucleic acids from bodily fluids (e.g., plasma).

We claim: 
     
         1 . A method of isolating nucleic acids, comprising:
 a) contacting a biological sample comprising cells with a lysis buffer comprising greater than 35% ethanol by volume;   b) contacting said sample of step a) with a solid support that binds cell-free nucleic acid wherein said solid support is a magnetic particle;   c) washing said solid support of step b) with a wash buffer; and   d) eluting nucleic acid from said solid support to generate isolated nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein said ethanol is present in said lysis buffer at a concentration of approximately 40% to 60%. 
     
     
         3 . The method of  claim 1 , wherein said ethanol is present in said lysis buffer at a concentration of approximately 45% to 55%. 
     
     
         4 . The method of  claim 1 , wherein said ethanol is present in said lysis buffer at a concentration of approximately 50%. 
     
     
         5 . The method of  claim 1 , wherein said nucleic acid is a circulating DNA. 
     
     
         6 . The method of  claim 5 , wherein said DNA is less than 1000 bases in length. 
     
     
         7 . The method of  claim 5 , wherein said DNA is less than 500 bases in length. 
     
     
         8 . The method of  claim 5 , wherein said DNA is less than 200 bases in length. 
     
     
         9 . The method of  claim 1 , wherein said sample is selected from the group consisting of blood, blood products, serum, and urine. 
     
     
         10 . The method of  claim 1 , wherein said sample is from a subject and the presence of said nucleic acid in said sample is indicative of a disease state in said subject. 
     
     
         11 . The method of  claim 10 , wherein said disease state is cancer. 
     
     
         12 . The method of  claim 1 , further comprising the step of analyzing said sample for the presence of said nucleic acid. 
     
     
         13 . The method of  claim 12 , wherein said analyzing comprises performing a nucleic acid detection assay selected from the group consisting of an amplification assay, a hybridization assay, a methylation status detection assay, and a sequencing assay. 
     
     
         14 . The method of  claim 13 , wherein said amplification is real time PCR and said methylation status detection assay is methylation specific PCR or heavy methyl PCR. 
     
     
         15 . A composition, comprising: a circulating DNA; a magnetic particle; and a lysis buffer comprising 35% or more ethanol by volume in a mixture with a biological sample comprising one or more cells. 
     
     
         16 . The composition of  claim 15 , wherein said ethanol is present in said lysis buffer at a concentration of approximately 45% to 55%.