Method of isolating nucleic acids for long sequencing reads

Abstract

The invention is a novel method of isolating long nucleic acids from samples suitable for nucleic acid sequencing. The method is especially suitable for isolating low-concentration nucleic acids, e.g., viral nucleic acids, from clinical samples.

1 . A method of isolating viral nucleic acids from a subject's blood plasma comprising
 (a) providing a blood plasma sample;   (b) subjecting the sample to a treatment with a detergent and a protease;   (c) obtaining a precipitate from the sample;   (d) contacting the precipitate with a buffer comprising an alcohol;   (e) contacting the sample from step (d) to a nucleic acid-retaining separation matrix to bind the nucleic acid;   (f) eluting the nucleic acid from the matrix with an elution buffer to obtain an elution volume;   (g) pooling at least two elution volumes from the same sample into a pooled elution volume; and   (h) concentrating the pooled elution volume at least 2-fold by passing the pooled elution volume through a semipermeable polymer membrane.   
     
     
         2 . The method of  claim 1 , wherein the treatment with the detergent and a protease is at a temperature of 56° C. or less. 
     
     
         3 . The method of  claim 1 , wherein the viral nucleic acid is between 1,000 and 10,000 nucleotides long. 
     
     
         4 . The method of  claim 1 , wherein the semipermeable polymer membrane is selected from a group consisting of cellulose acetate, nylon, polyether, and polyether sulfone. 
     
     
         5 . The method of  claim 1 , further comprising prior to step (f), treating the nucleic acids bound to the matrix with a nuclease. 
     
     
         6 . The method of  claim 5 , wherein the viral nucleic acid is DNA and the nuclease is RNase. 
     
     
         7 . The method of  claim 5 , wherein the viral nucleic acid is RNA and the nuclease is DNase. 
     
     
         8 . The method of  claim 1 , wherein the treatment in step (b) is in a lysis buffer comprising a detergent and one or more of high ionic strength salt, a reducing agent and a chaotropic agent. 
     
     
         9 . A method of isolating viral nucleic acids from a subject's blood plasma comprising
 (a) providing a blood plasma sample;   (b) subjecting the sample to a treatment with a detergent and a protease;   (c) obtaining a precipitate from the sample;   (d) contacting the precipitate with a buffer comprising an alcohol;   (e) contacting the sample from step (d) to a nucleic acid-retaining separation matrix to bind the nucleic acid;   (f) eluting the nucleic acid from the matrix with an elution buffer to obtain an elution volume;   (g) concentrating the pooled elution volume at least 2-fold by passing the pooled elution volume through a semipermeable polymer membrane.   
     
     
         10 . A method of detecting HIV in a subject's blood plasma comprising
 (a) providing a blood plasma sample;   (b) subjecting the sample to a treatment with a detergent and a protease;   (c) in the presence of alcohol, binding the nucleic acids in the sample to a nucleic acid-retaining separation matrix;   (d) eluting the nucleic acid from the matrix with an elution buffer to obtain an elution volume;   (e) pooling at least two elution volumes from the same sample into a pooled elution volume;   (f) concentrating the pooled elution volume at least 2-fold by passing the pooled elution volume through a semipermeable polymer membrane; and   (g) detecting the HIV nucleic acid in the concentrated elution volume wherein the limit of detection is 100 copies of the HIV nucleic acid in 1 ml of the plasma sample.   
     
     
         11 . The method of  claim 10 , wherein the detecting is by measuring concentration of the nucleic acid in the concentrated elution volume. 
     
     
         12 . The method of  claim 10 , wherein the detecting is by forming amplicons of the HIV nucleic acid. 
     
     
         13 . A method of detecting HCV in a subject's blood plasma comprising
 (a) providing a blood plasma sample;   (b) subjecting the sample to a treatment with a detergent and a protease;   (c) in the presence of alcohol, binding the nucleic acids in the sample to a nucleic acid-retaining separation matrix;   (d) eluting the nucleic acid from the matrix with an elution buffer to obtain an elution volume;   (e) pooling at least two elution volumes from the same sample into a pooled elution volume;   (f) concentrating the pooled elution volume at least 2-fold by passing the pooled elution volume through a semipermeable polymer membrane; and   (g) detecting the HCV nucleic acid in the concentrated elution volume wherein the limit of detection is 1000 IU of HCV in 1 ml of the plasma sample.   
     
     
         14 . The method of  claim 13 , wherein the detecting is by measuring concentration of the nucleic acid in the concentrated elution volume. 
     
     
         15 . The method of  claim 13 , wherein the detecting is by forming amplicons of the HIV nucleic acid.