Patent Application: US-201313918979-A

Abstract:
the present invention provides a chromatography column and a method for isolating nucleic acid molecules . in one embodiment , the present invention provides a double - layer column of a first anion exchange membrane and a second serially coupled silica membrane . upon flowing a nucleic acid - containing solution through the first anion exchange membrane , the nucleic acid binds to and then elutes from the first membrane . the eluted solution then flows serially through the second silica membrane , which the nucleic acid binds to and then elutes from . due to this novel serial coupled double - layer principle , the present invention is particularly suitable for co - isolating rna and dna , for isolating nucleic acid embraced by proteins , e . g ., viruses , and for isolating diluted nucleic acid in a large volume , e . g ., plasma . in addition , the eluted nucleic acid is ready for downstream applications .

Description:
in one aspect , the present invention is a double - layer chromatography column for isolating nucleic acid . it comprises at least two different solid layers of the first anion exchange medium , e . g ., a deae anion exchange membrane , and the second silica medium , e . g ., a silica membrane ( fig1 ). solutions flow through the first layer and then through the second layer . when a first solution containing nucleic acid flows through the first layer , the nucleic acid becomes bound ( fig2 ). when a second solution flows through the first layer to which the nucleic acid is bound , the bound nucleic acid is eluted . when it flows through the second layer , the eluted nucleic acid becomes bound . in this sense , the second solution functions as a “ serial coupler ”, i . e ., eluting the nucleic acid from the first layer and then binding the nucleic acid to the second layer . when a third solution flows through the second layer to which the nucleic acid is bound , the nucleic acid becomes eluted . the anion exchange membrane or bead comprises a supporting matrix and a covalently attached anion exchanger . weak anion exchanger of diethylamine ( d , or deae ), strong anion exchanger of quaternary ammonium ( q ), and another weak anion exchanger of diethylaminopropyl ( anx ) can be used ( table 1 ). many types of anion exchange bead and membrane are available , including waterman deae anion exchange cellulose membrane ( de81 paper ), vivapure deae anion exchange regenerated cellulose membrane used in vivapure iex d spin column , vivapure q anion exchange regenerated cellulose membrane used in vivapure iex q spin column , pall mustang q anion exchange cross - linked polymeric membrane used in acrodisc chromatography unit , pall deae ceramic hyperd f bead used in acrosep column , qiagen deae anion exchange bead with supporting matrix of silica bead , ge deae sepharose bead , ge q sepharose bead , and ge anx sepharose bead . we tested waterman deae anion exchange cellulose membrane ( de81 paper ). the binding principle is based on the interaction between the negatively charged phosphates of the dna backbone and the positively charged group , e . g ., deae , on the surface . the salt concentration and ph value of a solution used determine whether dna is bound or eluted . dna is bound to the anion exchanger surface in the presence of a low salt concentration at a low ph value , e . g ., 10 mm tris - hcl , 1 mm edta , ph 7 . 5 , which was used as the first solution in tested examples . dna is eluted in the presence of a high salt concentration at a high ph value , e . g ., 4 m gutc , 0 . 5 m nacl , 20 mm tris - hcl , ph 7 , which was used as the second solution in tested examples . the silica membrane or bead can be made of controlled pore glass , filters embedded with silica particles , silica gel particles , resins comprising silica in the form of diatomaceous earth , and glass fibers . we tested a silica membrane from ezgene plasmid purification miniprep kit of bioland . other examples include silica bead and membrane used in pureyield plasmid midiprep system from promega , wizard plus minipreps dna purification system from promega , and qiaprep miniprep kit from qiagen . the binding principle is based on the high affinity of the negatively charged dna backbone to the positively charged silica surface under concentrated chaotrophic salt conditions , e . g ., 4 m gutc , 0 . 5 m nacl , 20 mm tris - hcl , ph 7 , which was used as the second solution in tested examples . dna is eluted in the presence of a high salt concentration at a high ph value , e . g ., 10 mm tris - hcl , 0 . 1 mm edta , ph 9 . 0 , which was used as the third solution in tested examples . the first aqueous solution facilitates the nucleic acid to bind to the first layer . a solution of a low salt concentration at a low ph value , e . g ., 10 mm tris - hcl , 1 mm edta , ph 7 . 5 , may be used . the second aqueous solution facilitates the nucleic acid to be eluted from the first layer and to be bound to the second layer . it may contain 3 m to 6 m gutc , 20 mm tris - hcl , ph 6 . 0 - 7 . 5 . other chaotropic salts , e . g ., guhcl , nai , and nacio 4 , can also be used . in addition , chaotropic salts can be used in the presence of organic solvents , e . g ., ethanol or isopropanol . furthermore , the second solution can also comprise other salts , like nacl and kcl , to increase ion strength , e . g ., 4 m gutc , 0 . 5 m nacl , 20 mm tris - hcl , ph 7 . the third aqueous solution facilitates the nucleic acid to be eluted from the second layer . a solution of a low salt concentration at a high ph value , e . g ., 10 mm tris - hcl , 0 . 1 mm edta , ph 8 . 0 - 9 . 0 , may be used . in addition , between the first solution and the second solution , an aqueous solution bc can flow for washing the first layer that may comprise the same components as the first solution except for the nucleic acid , e . g ., 10 mm tris - hcl , 1 mm edta , ph 7 . 5 , which was used in tested example . furthermore , between the second solution and the third solution , an aqueous solution cd can flow for washing the second layer . the solution cd can be composed of the same as the second solution , or the solution cd can be made up differently , e . g ., 10 mm tris - hcl , 80 % ethanol , ph 7 . 5 , which was used in tested examples . we tested 1 kb dna marker and human genomic dna . however , the nucleic acid ( s ) may be dna and rna in any possible configuration , e . g ., in the forms of linear , circular , double - stranded , and single - stranded . the nucleic acid ( s ) may be contained in a particle , e . g ., virus . in this case , the ph value of the first solution containing the virus can be adjusted to make the embraced proteins negatively charged , and thus the proteins is bound to the first layer . driven by centrifuge or vacuum , a little volume of the first solution is retained on and within the first layer . when the second solution containing as high as 6 m gutc is added to the first layer , the resulting guanidinium salt will not be diluted too much by the little volume of the retained first solution . therefore , the concentrated guanidinium salt is able to lyse the particle on the first layer , making the nucleic acid released . thus , the first layer functions as a “ binder ”, a “ concentrator ” and a “ lyser ”. dna and rna can be isolated together due to the “ binder ” and “ concentrator ” functions of the first layer . the resulting high concentration of guanidinium salt can make the dna and rna efficiently bound to the second layer . a little amount of nucleic acid in a large volume of sample , e . g ., plasma , can be isolated due to the “ binder ” and “ concentrator ” functions of the first layer , as described above . 1 kb dna marker was from biolabs that contains 10 double - stranded dna fragments of 0 . 5 , 1 , 1 . 5 , 2 , 3 , 4 , 5 , 6 , 8 , and 10 kilobases ( kb ). the silica membrane column was from bioland ezgene plasmid purification miniprep kit . whatman de81 cellulose chromatography paper , a deae membrane , was from ge healthcare . genomic dna was extracted from blood white cells and stored in 10 mm tris - hcl ( ph 8 . 0 ) and 1 mm edta buffer using dneasy mini blood and tissue kit according to the manufacturer &# 39 ; s protocol ( qiagen ). the concentration was measured by a spectrophotometer at 260 nm . it was stored at − 20 ° c . until used . whatman de81 paper was cut into disks with diameter of 7 mm . five such disks were assembled and put on the top of the silica membrane that was already installed in the spin column ( fig3 ). thus , the double - layer column was prepared and used for isolating nucleic acid . using the double - layer column , we tested the human genomic dna and lkb dna marker . 200 μl of the first solution ( 10 mm tris - hcl , 1 mm edta , ph 7 . 5 ) containing 2 . 5 μg of the 1 kb dna marker and 2 . 5 μg of genomic dna was transferred into each column and centrifuged at 8 , 000 rpm for 2 minutes . the filtrate was discarded . for washing , 400 μl of 1 × te buffer ( 10 mm tris - hcl , 1 mm edta , ph 7 . 5 ) was added into each column and centrifuged at 10 , 000 rpm for 1 minute . the filtrate was discarded . 600 μl of the second solution was added into each column . four guanidine salt conditions were examined : 1 ) 4 m gutc , 0 . 5 m nacl , 20 mm tris - hcl , ph 7 ; 2 ) 3 . 6 m gutc , 1 m nacl , 20 mm tris - hcl , ph 7 ; 3 ) 3 m gutc , 2 m nacl , 20 mm tris - hcl , ph 7 ; and 4 ) 3 . 6 m guhcl , 0 . 5 m nacl , 20 mm tris - hcl , ph 7 . the columns were centrifuged at 10 , 000 rpm for 1 minute . the filtrate was discarded for washing , 600 μl of wash buffer ( 10 mm tris - hcl , 80 % ethanol , ph 7 . 5 ) was added into each column and centrifuged at 10 , 000 rpm for 1 minute . the filtrate was discarded . 100 μl of the third solution ( 10 mm tris - hcl , 0 . 1 mm edta , ph 9 . 0 ) was added into each column and centrifuged at 13 , 000 rpm for 1 minute . the filtrate was collected as output . the concentration of the collected dna in te buffer ( 10 mm tris - hcl , 0 . 1 mm edta , ph 9 . 0 ) was measured by a spectrophotometer at 260 nm and 280 nm ( table 2 ). with 4 m gutc , 0 . 5 m nacl , 20 mm tris - hcl , ph 7 , as the second solution , the total amount of dna was collected the most , showing the effect of guanidine salt conditions . b it contained the 1 kb dna marker and genomic dna collected in 100 μl of the third solution , and its total amount was determined by the spectrophotometer at 260 nm . c the four guanidine salt conditions were 1 ) 4m gutc , 0 . 5m nacl , 20 mm tris - hcl , ph 7 ; 2 ) 3 . 6m gutc , 1m nacl , 20 mm tris - hcl , ph 7 ; 3 ) 3m gutc , 2m nacl , 20 mm tris - hcl , ph 7 ; and 4 ) 3 . 6m guhcl , 0 . 5m nacl , 20 mm tris - hcl , ph 7 . 50 μl of the collected dna sample eluted from the third solution was electrophoresed through a standard 1 % agarose gel ( fig4 ). for comparison , the filtrates of the first solution ( 50 % of the amount ) and the filtrates of 1 × te washing buffer ( 50 % of the amount ) were also loaded . the gel was stained with ethidium bromide for uv photography by a charge - coupled device camera . compared with the band patterns of the untreated 1 kb dna marker and genomic dna on the agarose gel , the treated dna bands showed consistent intensities under each of the four guanidine salt conditions , suggesting high quality . in addition , the filtrates of the first solution and the filtrates of 1 × te washing buffer did not show any observable bands , suggesting the 1 kb dna marker and genomic dna in the first solution were bound to the whatman de81 paper and could not be washed away by the 1 × te washing buffer . furthermore , to show the necessity of the deae membrane in the double - layer column , a column only with the silica membrane ( single - layer ) was tested under the same conditions . there was virtually no any dna collected in the third solution . thus , both layers are needed for the isolation . a for example , p53 means the human p53 gene ; ( 13280 ), 5 ′ end of the primer begins at nucleotide 13280 according to genbank accession : x54156 ; d , downstream ( i . e ., in the direction of transcription ). the precise sizes and locations of the pcr fragment can be obtained from the informative names . the primers were designed to amplify a 209 - bp region of exon 6 of the p53 gene . each pcr mixture contained a total volume of 25 μl : 50 mm kcl , 10 mm tris - hcl ( ph 8 . 3 ), 1 . 5 mm mgcl 2 , 200 μm each dntp , 0 . 1 μm each primer for the human p53 gene , 0 . 1 × sybr green i dye , 0 . 02 % twee - 20 , 0 . 02 % np - 40 , 50 ng of total dna , and 1 u of taq polymerase . a bio - rad cfx96 real time pcr detection system was used for quantification of the amplified product . analysis mode : sybrgreen fluorophore , baseline setting : baseline subtracted curve fit , threshold cycle ( ct ) determination : single threshold , baseline method : sybr auto calculated , threshold setting : auto calculated . the cycling entailed denaturation at 94 ° c . for 15 seconds , annealing at 55 ° c . for 30 seconds , and elongation at 72 ° c . for 1 minute for 30 cycles . before thermocycling , a step of 94 ° c . for 2 minutes was applied to completely denature the genomic dna . after the last cycle , melting curving analysis was followed from 68 ° c . to 95 ° c . with increment 0 . 5 ° c . and holding 5 seconds . we examined the untreated and the four differently treated dna samples using real - 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