Preparative electrophoretic method for targeted purification of genomic dna fragments

Abstract

A sample containing particles having high-molecular-weight (HMW) DNA is entrapped in a gel matrix, and the gel matrix is exposed to a lysis reagent configured to release the HMW DNA from the particles. The HMW DNA may be purified by subjecting the gel matrix to an electrophoretic field that removes the HMW DNA from the particles, lysis reagents, and/or other sample constituents, from the gel matrix such that the HMW DNA remains. The gel matrix may be subjected with DNA cleavase re-agents configured to cleave at specific DNA sequences within the HMW DNA to liberate defined segments of the DNA as fragments of reduced size. The gel matrix may also be subjected to an electrophoretic field, which moves and separates the DNA fragments from uncleaved DNA of the HMW DNA, which remains substantially immobile. The electrophoretically separated DNA fragments may be isolated from the gel matrix.

We claim: 
     
         1 . A method for isolating fragments of genomic DNA comprising:
 providing a sample containing particles having high-molecular-weight (HMW) DNA;   entrapping the sample in a gel matrix;   exposing the gel matrix with the entrapped sample to a lysis reagent, the lysis reagent configured to release the HMW DNA from the particles;   purifying the HMW DNA of the sample by subjecting the gel matrix to an electrophoretic field configured to remove the HMW DNA from the particles, lysis reagents, and/or other sample constituents, from the gel matrix such that the BMW DNA remains;   subjecting the gel matrix with DNA cleavase reagents configured to cleave at specific DNA sequences within the BMW DNA so as to liberate defined segments of the DNA as fragments of reduced size;   subjecting the gel matrix to an electrophoretic field, the field being configured to:
 move and thereby separate the DNA fragments from uncleaved DNA of the HMW DNA which remains substantially immobile; and 
   isolating the electrophoretically separated DNA fragments from the gel matrix.   
     
     
         2 . The method of  claim 1 , wherein the uncleaved remainder of the HMW DNA remains entrapped in the gel matrix. 
     
     
         3 . The method of any proceeding claim, wherein the particles are contained in a liquid suspension and comprise at least one of intact cells selected from the group consisting of: animal, plant, bacterial, fungal, archebacterial, protozoan, and intact virus particles. 
     
     
         4 . The method of any proceeding claim, wherein the gel matrix comprises an agarose hydrogel at a concentration between about 0.2% and about 5% (weight/volume). 
     
     
         5 . The method of any proceeding claim, wherein the lysis reagent comprises an anionic detergent at a concentration between 0.05% and 10%. 
     
     
         6 . The method of  claim 5 , wherein the anionic detergent is sodium dodecyl sulfate (SDS). 
     
     
         7 . The method of any proceeding claim, wherein the size of the BMW DNA is >10 megabase pairs in length, and the size of the DNA fragments is <2 megabase pairs in length. 
     
     
         8 . The method of  claim 3 , wherein upon the particles comprising bacteria, plant, or fungal cells, the method further comprises subjecting the gel matrix to other enzymatic reagent treatments configured to remove the cell walls prior to lysis. 
     
     
         9 . The method of any proceeding claim, wherein the DNA fragments are isolated from the gel matrix by electroelution into an elution module containing liquid buffer. 
     
     
         10 . The method of any proceeding claim, wherein DNA cleavase reagents comprise one or more RNA-guided endonuclease compositions. 
     
     
         11 . The method of  claim 11 , wherein the RNA-guided endonuclease composition is based on the Cas9 protein with guide RNAs configured to enable the guide-RNA-Cas9 complex to cleave at specific user-designated sites of the HMW DNA. 
     
     
         12 . A method for isolating fragments of genomic DNA comprising:
 providing a sample containing high-molecular-weight (HMW) DNA;   entrapping the sample in a gel matrix;   subjecting the gel matrix with DNA cleavase reagents configured to cleave at specific DNA sequences within the BMW DNA so as to liberate defined segments of the HMW DNA as fragments of reduced size;   subjecting the gel matrix to an electrophoretic field, the field being configured to:
 move and thereby separate the DNA fragments from uncleaved DNA of the HMW DNA which remains substantially immobile; and 
   isolating the electrophoretically separated DNA fragments from the gel matrix.   
     
     
         13 . A method for isolating fragments of genomic DNA comprising:
 entrapping high-molecular-weight (HMW) DNA in a gel matrix;   subjecting the gel matrix with DNA cleavase reagents configured to cleave at specific DNA sequences within the HMW DNA so as to liberate defined segments of the HMW DNA as fragments of reduced size;   subjecting the gel matrix to an electrophoretic field, the field being configured to:
 move and thereby separate the DNA fragments from uncleaved DNA of the HMW DNA which remains substantially immobile; and 
   isolating the electrophoretically separated DNA fragments from the gel matrix.   
     
     
         14 . An apparatus or system for performing the method according to any of  claims 1 - 13 .