Nucleic Acid Extraction from Heterogeneous Biological Materials

Abstract

Methods for extracting high quality nucleic acids from a heterogenous collection of nucleic acid-containing materials from a biological sample are disclosed. The heterogenous collection of nucleic-acid containing materials may contain cells or microvesicles, or both. The extractions obtained by the methods described herein are characterized by high yield and high integrity, making the extracted nucleic acids useful for various applications in which high quality nucleic acid extractions are preferred, e.g., a diagnosis, prognosis, or therapy evaluation for a medical condition.

1 . A method of extracting nucleic acid from a biological sample, comprising the steps of:
 a) obtaining a biological sample;   b) performing a sample pre-processing step on the biological sample to obtain a fraction comprising a heterogeneous collection of nucleic acid-containing materials, wherein the heterogeneous collection of nucleic acid-containing materials comprises cells or microvesicles, or both;   c) performing an operation selected from
 i) an extraction enhancement operation; 
 ii) an affinity exclusion operation; and 
 iii) an extraction enhancement operation and an affinity exclusion operation; and 
   d) extracting nucleic acid from the resulting materials.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein a marker listed in Table 1 or Table 2 is used in the affinity exclusion operation. 
     
     
         5 . The method of  claim 1 , further comprising an affinity enrichment operation. 
     
     
         6 . The method of  claim 5 , wherein a marker listed in Table 1 or Table 2 is used in the affinity enrichment operation. 
     
     
         7 . The method of  claim 1 , wherein the biological sample is a bodily fluid. 
     
     
         8 . The method of  claim 7 , wherein the bodily fluid is urine. 
     
     
         9 . The method of  claim 7 , wherein the bodily fluid is serum or plasma. 
     
     
         10 . The method of  claim 1 , wherein the extraction enhancement operation comprises:
 a) the addition of one or more of the following agents to the biological sample:
 (i) RNase inhibitor; 
 (ii) protease; 
 (iii) reducing agent; 
 (iv) decoy substrate, such as synthetic RNA; 
 (v) soluble receptor; 
 (vi) small interfering RNA, 
 (vii) RNA binding molecule, such as anti-RNA antibody, chaperone protein, or an RNase inhibitory protein; 
 (viii) RNase denaturing substance, such as high osmolarity solution, detergent; or 
   b) the performance of one or more of the following steps prior to nucleic acid extraction:
 (ix) washing; 
 (x) size-separating RNase from the sample; 
 (xi) effecting RNase denaturation through a physical change, such as by decreasing temperature, freeze/thaw cycle; or 
   c) any combination of the foregoing agents or steps.   
     
     
         11 . The method of  claim 1 , wherein the extraction enhancement operation comprises the addition of an RNase inhibitor. 
     
     
         12 . The method of  claim 11 , wherein the RNase inhibitor has a concentration of greater than [1×] concentration; alternatively, greater than or equal to [5×] concentration; alternatively, greater than or equal to [10×] concentration; alternatively, greater than or equal to [25×] concentration; and alternatively, greater than or equal to [50×] concentration. 
     
     
         13 . The method of  claim 11 , wherein the RNase inhibitor is a protease. 
     
     
         14 . A nucleic acid extraction from a heterogeneous collection of nucleic acid-containing materials taken from a biological sample, wherein the heterogeneous collection of nucleic acid-containing materials comprise cells or microvesicles, and wherein the quantitative ratio of 18S rRNA to 28S rRNA detectable in the extraction is within the range of approximately 1:1 to approximately 1:2; and is preferably approximately 1:2. 
     
     
         15 . A nucleic acid extraction from a heterogeneous collection of nucleic acid-containing materials taken from a biological sample, wherein the heterogeneous collection of nucleic acid-containing materials comprise cells or microvesicles, and wherein the nucleic acid yield is greater than or equal to 50 pg/ml from 1 ml or 20 ml of biological sample. 
     
     
         16 . (canceled) 
     
     
         17 . A kit for extracting nucleic acid from a biological sample, comprising:
 a) an affinity agent;   b) an extraction enhancement agent; and   c) a lysis buffer.   
     
     
         18 . The kit of  claim 17 , wherein the biological sample is a heterogeneous collection of nucleic acid-containing materials, wherein the heterogeneous collection of nucleic acid-containing materials comprise cells or microvesicles, or both
 c).   
     
     
         19 . The kit of  claim 17 , wherein the affinity agent is capable of binding to one or more markers listed in Table 1 or Table 2. 
     
     
         20 . The kit of  claim 19 , further comprising DNase, RNase, or both. 
     
     
         21 . The kit of  claim 19 , further comprising instructions for using the kit located either in the package with the other kit components or in a different location accessible to a kit user. 
     
     
         22 . The kit of  claim 19 , wherein the extraction enhancement agent is selected from the group consisting of:
 a) RNase inhibitor;   b) protease;   c) reducing agent;   d) decoy substrate;   e) soluble receptor;   f) small interfering RNA;   g) RNA binding molecule;   h) RNase denaturing substance; or   i) a combination of any of the foregoing.   
     
     
         23 . A kit for extracting nucleic acid from a biological sample, comprising:
 a) an affinity agent;   b) optionally, a lysis buffer or an extraction enhancement agent; and   c) instructions for using the kit, located either in the package with the other kit components or in a different location accessible to a kit user, wherein the instructions comprise instructions for using the affinity agent in an affinity exclusion operation.   
     
     
         24 . The kit of  claim 23 , wherein the biological sample is a heterogeneous collection of nucleic acid-containing materials, wherein the heterogeneous collection of nucleic acid-containing material comprise cells or microvesicles, or both
 c).   
     
     
         25 . The kit of  claim 23 , further comprising a second affinity agent, and instructions for using the second affinity agent in an affinity enrichment operation. 
     
     
         26 . The kit of  claim 23 , further comprising an extraction enhancement agent, and instructions for its use. 
     
     
         27 . The kit of  claim 23 , further comprising DNase, RNase, or both and instructions for their use. 
     
     
         28 . The use of the nucleic acid extraction method of  claim 1  in patient diagnostics, prognostics, theranostics, monitoring, predictive medicine, personalized medicine, integrated medicine, pharmacodiagnostics or companion diagnostics. 
     
     
         29 . The use of  claim 28 , wherein the nucleic acid obtained from practice of the extraction method is analyzed for the presence or absence of a genetic aberration associated with a disease or other medical condition. 
     
     
         30 . The method of  claim 1 , wherein the heterogeneous collection of nucleic acid-containing materials comprises cells or microvesicles, or both.