Methods of Nucleic Acid Fractionation and Detection

Abstract

The invention provides methods of detecting a nucleic acid present in a biological sample, comprising combining the biological sample with a lysis buffer to form a lysis mixture comprising nucleic acid released from cells in said biological sample; and subjecting a volume of the lysis mixture to size-exclusion chromatography in a column comprising a volume of size-exclusion medium. In certain embodiments, the lysis buffer separates double-stranded nucleic acid into single-stranded nucleic acid. In certain embodiments, the elution can have a flow rate of separation of less than 10 minutes to produce an eluted solution comprising isolated nucleic acid. The invention provides for a method of accurately and rapidly detecting products of nucleic acid amplification.

1 .- 21 . (canceled) 
     
     
         22 . A method of purifying a nucleic acid from a biological sample, comprising the steps of:
 a) combining the biological sample with a lysis buffer to form a lysis mixture comprising nucleic acid released from cells in said biological sample;   b) applying a volume of the lysis mixture to size-exclusion chromatography medium in a column comprising a loading end, an eluting end, and a volume of size-exclusion medium, wherein said volume of lysis mixture is 0.35 to 0.8 of the volume of the size-exclusion medium, and allowing lysis mixture to completely enter the size-exclusion chromatography medium; and   c) providing a positive pressure differential to the loading side of the column forcing interstitial fluid containing nucleic acid from to drain from SEC medium, and collecting drained fluid containing nucleic acid for further amplification or analysis.   
     
     
         23 . The method of  claim 22 , wherein the lysis mixture enters the SEC medium by gravity flow. 
     
     
         24 . The method of  claim 22 , wherein said nucleic acid is DNA, RNA, or a mixture thereof. 
     
     
         25 . The method of  claim 22 , further comprising equilibrating said chromatography column with an equilibrating buffer comprising 1-10 mM Mg 2+  or a non-ionic detergent, or a combination, prior to the subjecting of step b). 
     
     
         26 . The method of  claim 22 , wherein said lysis buffer comprises alkali hydroxide at pH>11. 
     
     
         27 . The method of  claim 22 , wherein said lysis buffer comprises urea or chaotropic salt. 
     
     
         28 . The method of  claim 22 , wherein said lysis buffer separates double stranded nucleic acid into single stranded nucleic acid and inhibits nucleic acid interactions with protein. 
     
     
         29 . The method of  claim 22 , wherein said size-exclusion medium comprises polyacrylamide, polybisacrylamide or polymethacrylamide. 
     
     
         30 . The method of  claim 22 , wherein said nucleic acid is DNA, RNA, or a mixture thereof. 
     
     
         31 . The method of  claim 22 , further comprising the later step of analyzing said isolated nucleic acid using an enzyme-catalyzed reaction. 
     
     
         32 - 43 . (canceled)