Enrichment of cell-free dna from a biological sample

Abstract

Provided are methods and compositions for enriching cfDNA fragments from a biological fluid sample. A biological fluid sample, such as a urine sample, is collected and, in certain examples, pretreated before enrichment of the cfDNA. For the pretreatment, the sample is centrifuged to remove large cells and large cellular debris. As part of the pretreatment, the sample is also cleared of additional large cellular debris and excess volume by subjecting the sample to anion exchange chromatography and eluting bound DNA. Following any pretreatment of the sample, different concentrations an alcoholic solution are used—along with a mixture of DNA-binding particles and a chaotropic agent—to enrich the sample with cfDNA fragments having different sizes. For example, a biological sample can be enriched with small cfDNA fragments less than about 100 base pairs in length or large cfDNA fragments greater than about 100 base pairs in length.

1 . A method for enriching cell-free DNA (cfDNA) from a biological fluid sample, comprising:
 contacting a biological fluid sample with an alcoholic solution, a chaotropic agent, and a plurality of DNA-binding particles to form a reaction solution, wherein the reaction solution has an alcohol concentration of at least 45% v/v;   separating the plurality of DNA-binding particles from the reaction solution, thereby forming a particle fraction and a non-particle fraction from the reaction solution; and   eluting cfDNA fragments from the plurality of DNA-binding particles of the particle fraction, wherein the eluate is enriched with cfDNA fragments having a length of at least about 100 base pairs.   
     
     
         2 . The method of  claim 1 , wherein the DNA-binding particles are silica magnetic bead particles and wherein separating the plurality of silica particles from the reaction solution comprises subjecting the reaction solution to a magnetic field. 
     
     
         3 . The method of  claim 1 , wherein the concentration of alcohol in the reaction solution is at least 55% v/v. 
     
     
         4 . The method of  claim 1 , wherein the eluate is enriched with cfDNA fragments of about 160 base pairs in length, of about 300 base pairs in length, and of about 10,000 base pairs in length, or combinations thereof. 
     
     
         5 . The method of  claim 1 , wherein the alcohol of the alcoholic solution is isopropyl alcohol. 
     
     
         6 . The method of  claim 1 , wherein the biological fluid sample is a urine sample. 
     
     
         7 . The method of  claim 5 , wherein the cDNA fragments consist essentially of post-glomerular cfDNA fragments. 
     
     
         8 .- 16 . (canceled) 
     
     
         17 . A method for enriching a biological fluid sample with cell-free DNA (cfDNA), comprising:
 contacting a biological fluid sample with an alcoholic solution, a chaotropic agent, and a plurality of DNA-binding particles to form a reaction solution;   separating the plurality of DNA-binding particles from the reaction solution, thereby forming a particle fraction and a non-particle fraction from the reaction solution, wherein the particle fraction and non-particle particle fraction are enriched with cfDNA fragments having different size distributions.   
     
     
         18 . The method of  claim 17 , wherein the plurality of DNA-binding particles are silica magnetic bead particles and wherein separating the plurality of silica magnetic bead particles from the reaction solution comprises subjecting the reaction solution to a magnetic field. 
     
     
         19 . The method of  claim 18 , wherein the reaction solution has an alcohol concentration greater than about 45% v/v alcohol. 
     
     
         20 . The method of  claim 19 , further comprising eluting the cfDNA fragments from the plurality of bead particles of particle fraction. 
     
     
         21 . The method of  claim 20 , wherein the eluate is enriched with cfDNA fragments having a length of about 160 base pairs, about 300 base pairs, and about 10,000 base pairs, or combinations thereof. 
     
     
         22 . The method of  claim 17 , wherein the alcohol is isopropyl alcohol. 
     
     
         23 . The method of  claim 17 , wherein the biological sample is a urine sample. 
     
     
         24 . The method of  claim 23 , wherein the cfDNA fragments consist essentially of post-glomerular cfDNA fragments. 
     
     
         25 . The method of  claim 17 , wherein the reaction solution has an alcohol concentration less than about 35% v/v alcohol. 
     
     
         26 . The method of  claim 25 , further comprising:
 contacting the non-particle fraction with a second alcoholic solution and a second plurality of DNA-binding particles to form a second reaction solution, wherein the second reaction solution has an alcohol concentration greater than about 45% v/v alcohol; and   separating the second plurality DNA-binding particles from the second reaction solution; and,   eluting the cfDNA fragments from the second plurality of DNA-binding particles.   
     
     
         27 . The method of  claim 25 , wherein the second plurality of DNA-binding particles are silica magnetic bead particles and wherein separating the plurality of silica magnetic bead particles from the second reaction solution comprises subjecting the second reaction solution to a magnetic field. 
     
     
         28 . The method of  claim 27 , wherein the eluate is enriched with cfDNA fragments having a length of about 10 nucleotide base pairs to about 80 nucleotide base pairs. 
     
     
         29 . The method of  claim 28 , wherein the eluate is enriched with cfDNA fragments having a length of about 30 nucleotide base pairs. 
     
     
         30 .- 31 . (canceled)