Isolation of High Molecular Weight DNA Using Beads

Abstract

Provided herein is a method for isolating high molecular weight (HMW) DNA using beads that are at least 200 μm in diameter that utilizes a device for retaining the beads and where the purified DNA eluant exits the device without shearing the HMW DNA. In some embodiments, the method comprises precipitating the DNA onto the beads, washing the beads in the device, and then eluting the DNA from the beads therein while substantially avoiding shear. Compositions and kits for practicing the method are also provided.

What is claimed is: 
     
         1 . A kit comprising:
 (a) one or more beads that have a smallest dimension of 2 mm-6 mm; and   (b) a bead retaining tube having an opening for receiving a DNA preparation and one or more exit openings, wherein each exit opening has a smallest dimension that is at least 0.5 mm but smaller than the smallest dimension of the one or more beads, and wherein the bead retaining tube comprises:
 (i) at least one ridge, flange, collar, or other bead retaining structure for supporting the one or more beads above the one or more exit openings so that the one or more exit openings remain unblocked by the one or more beads; or 
 (ii) a series of exit openings arranged in a grid. 
   
     
     
         2 . The kit according to  claim 1 , wherein the one or more beads are formed of rough or smooth material having a surface charge; and/or wherein the one or more beads are formed from glass or are ceramic beads. 
     
     
         3 . The kit according to  claim 1 , wherein the one or more beads are spherical. 
     
     
         4 . The kit according to  claim 1 , wherein the one or more exit openings of the bead retaining tube are circular. 
     
     
         5 . The kit according to  claim 1 , comprising 1-50 spherical beads, wherein the number of beads in the kit is inversely proportional to the diameter of the beads; and wherein the bead retaining tube has a volume large enough to contain the beads. 
     
     
         6 . The kit according to  claim 5 , wherein:
 (i) the bead retaining tube is a 1-2 ml tube; and   (ii) the kit comprises:
 1 bead of 5 mm or 6 mm diameter, 
 2 beads of 4 mm diameter, 
 4 beads of 3 mm diameter, or 
 8 beads of 2 mm diameter. 
   
     
     
         7 . A composition comprising:
 (a) one or more beads having a smallest dimension of 2 mm-6 mm;   (b) a DNA preparation comprising a high molecular weight (HMW) DNA; and   (c) a DNA precipitant;   wherein the one or more beads, the DNA preparation, and the DNA precipitant are contained in a bead retaining tube, wherein the DNA adheres to the beads;   wherein the bead retaining tube has one or more outlet openings, wherein each outlet opening has a smallest dimension that is at least 0.5 mm but less than the smallest dimension of the one or more beads, and   wherein the bead retaining tube comprises: (i) at least one ridge, flange, collar, or other bead retaining structure for supporting the one or more beads above the one or more exit openings so that the one or more exit openings remain unblocked by the one or more beads; or (ii) a series of exit openings arranged in a grid.   
     
     
         8 . The composition according to  claim 7 , wherein:
 the one or more beads are spherical; optionally wherein the one or more spherical beads are 1-50 spherical beads and/or wherein the one or more spherical beads are spherical glass beads.   
     
     
         9 . The composition according to  claim 7 , wherein the DNA preparation is obtained from a cell lysate, such as wherein the DNA preparation is the product of a cleanup reaction from a cell lysate; or wherein the DNA preparation is a cell lysate or a tissue lysate. 
     
     
         10 . The composition according to  claim 1 , wherein the DNA precipitant is selected from an alcohol, a chaotropic salt and a crowding agent. 
     
     
         11 . A tube comprising:
 (a) a wall, the wall having a first end defining a first opening and a second end defining at least one exit opening, wherein the diameter of the first end is larger than the diameter of the exit opening and wherein the exit opening has a diameter smaller than the one or more beads and the beads do not block the at least one exit opening;   (b) one or more beads, each defined by a surface and having a diameter of at least 1 mm; and   (c) DNA adhered to the surface of the one or more beads.   wherein the wall encircles the one or more beads.   
     
     
         12 . The tube of  claim 11 , wherein the second end defining at least one exit opening further defines a plurality of exit openings, each exit opening having a diameter smaller than the one or more beads. 
     
     
         13 . The tube of  claim 11 , wherein the second end further comprises a collar comprising at least one ridge, the at least one ridge contacting at least one of the one or more beads and preventing the one or more beads from blocking the at least one exit opening. 
     
     
         14 . The tube of  claim 11 , wherein the beads have a charged surface. 
     
     
         15 . The tube of  claim 11 , wherein the beads are spherical. 
     
     
         16 . The tube of  claim 11 , wherein the beads are glass beads or ceramic beads. 
     
     
         17 . The tube of  claim 11 , wherein the beads have a diameter of up to 6 mm. 
     
     
         18 . The tube of  claim 11 , wherein the DNA has a median length of at least 50 kb or at least 50 kb 200 kb or at least 500 kb or at least 1 Mb. 
     
     
         19 . The tube of  claim 11 , wherein the DNA has a median length of at least 50 kb or at least 50 kb 200 kb or at least 500 kb or at least 1 Mb. 
     
     
         20 . The tube of  claim 11 , wherein the tube is sized to fit within an outer tube, the outer tube having a volume of 1 mL to 2 mL. 
     
     
         21 . A tube comprising a wall, the wall:
 having a first end defining a first opening and a second end defining at least one exit opening, wherein the diameter of the first end is larger than the diameter of the exit opening and wherein the exit opening has a diameter smaller than the one or more beads and the beads do not block the at least one exit opening; and   enclosing (a) one or more beads, each defined by a surface and having a diameter of at least 1 mm, and (b) DNA adhered to the surface of the one or more beads.   
     
     
         22 . A tube comprising a wall, the wall having a first end defining a first opening and a second end defining at least one exit opening, the tube containing (a) one or more beads, each defined by a surface and having a diameter of at least 1 mm, and (b) DNA adhered to the surface of the one or more beads, wherein the exit opening has a diameter smaller than the one or more beads and the beads do not block the at least one exit opening.