Apparatus and method for separating and purifying polynucleotides

Abstract

A method for removing a target DNA fragment having a predetermined base-pair length from a mixture of DNA fragments comprises the following steps. A mixture of DNA fragments which may contain the target DNA fragments is applied to a separation column containing media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent. The target DNA fragments are separated from the media by contacting it with a second solvent solution containing a counterion and a concentration of driving solvent in cosolvent which has been predetermined to remove DNA fragments having the target DNA fragment base pair length from the media. The target DNA fragments can be collected and optionally amplified. When the method is being applied to collect a putative fragment, if present, no DNA fragments having the base pair length of the target DNA could be present in the mixture. Alternatively, DNA fragments having the base pair length of the target DNA are present in the mixture. The disclosure also describes an ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprises a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein. The separation media has nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media.

The invention claimed is:  
     
         1 . A method for isolating targeted DNA fragments having a predetermined base-pair length from a mixture of DNA fragments comprising: 
 a) applying the mixture of DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solution containing counterion and a DNA binding concentration of driving solvent; and    b) removing the targeted DNA fragments from the media by contacting the media with a second solution containing counterion and a concentration of driving solvent which has been predetermined to remove DNA fragments having the targeted fragments from the separation media into a distinct segment of eluant.    
     
     
         2 . A method of  claim 1  including the step of: 
 c) collecting the distinct segment of eluant, whereby the targeted DNA fragments are recovered.  
 
     
     
         3 . A method of  claim 2  wherein the recovered targeted DNA fragments are amplified.  
     
     
         4 . A method of  claim 2  wherein the recovered targeted DNA fragments are cloned.  
     
     
         5 . A method for determining the presence of DNA fragments having a specific base-pair length in a sample mixture comprising: 
 d) applying the sample mixture to a separation column containing separation media having a nonpolar, nonporous surface, the sample mixture being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent; and    e) removing any DNA in the sample having said specific base pair length by contacting the separating media with a second solvent solution containing a counterion and a concentration of driving solvent which has been predetermined to remove DNA fragments having said base pair from the separation media.    
     
     
         6 . A method of  claim 5  wherein the second solvent solution produced in step (b) is collected.  
     
     
         7 . A method of  claim 6  wherein the second solvent solution product of step(b) is analyzed to determine whether or not DNA fragments having said base pair length are present in the sample mixture.  
     
     
         8 . A method of  claim 7  wherein any DNA in the second solvent solution product of step (b) is amplified.  
     
     
         9 . A method of  claim 8  wherein DNA fragments having said base pair length are present in the sample mixture in a concentration which is too low to be detected in the analysis, and the amplified product is analyzed to verify the presence of DNA fragments having said base pair length in the sample mixture.  
     
     
         10 . A method for separating by DMIPC target homoduplex and/or heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of: 
 a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent; and    b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site thereof, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.    
     
     
         11 . A method of  claim 10  wherein the desired fragments are a homoduplex fraction, the concentration of driving solvent is selected to remove one or both of the homoduplex fractions as separate fractions from homoduplex fractions.  
     
     
         12 . A method of  claim 11  including the step of collecting a homoduplex fraction.  
     
     
         13 . A method of  claim 12  including amplifying the collected homoduplex fraction.  
     
     
         14 . A method of  claim 10  wherein the desired fragments are a heteroduplex fraction, the concentration of driving solvent is selected to remove one or both of the heteroduplex fractions as separate fractions from homoduplex fractions.  
     
     
         15 . A method of  claim 11  including the step of collecting a heteroduplex fraction.  
     
     
         16 . A method of  claim 12  including amplifying the collected heteroduplex fraction.  
     
     
         17 . A method of  claim 10  wherein the ratio of the amounts of the heteroduplex fragments to homoduplex fragments is less than 1:1.  
     
     
         18 . A method of  claim 17  wherein the amount of heteroduplex in the separate heteroduplex fraction is below the level of detection.  
     
     
         19 . A method of  claim 10  wherein the retention time used in the separation of the homoduplex or heteroduplex fraction was previously determined from a reference standard.  
     
     
         20 . A method of  claim 19  wherein the reference standard was obtained by separating a standard mixture of homoduplex and heteroduplex, having the same base pair sequence as the sample, by Matched Ion Polynucleotide Chromatography.  
     
     
         21 . A method for determining the presence of a homoduplex or a heteroduplex faction in a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of: 
 a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent; and    b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site thereof, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.    
     
     
         22 . A method of  claim 21  including the step of: 
 c) collecting the distinct segment of eluant, whereby the targeted DNA fragments are recovered.  
 
     
     
         23 . A method of  claim 22  wherein the recovered targeted DNA fragments are amplified.  
     
     
         24 . A method of  claim 22  wherein the recovered targeted DNA fragments are cloned.  
     
     
         25 . A method of  claim 22  wherein said DNA sample contains a large background of wild type.  
     
     
         26 . A method of  claim 22  wherein said mutant DNA is below the limit of detection.  
     
     
         27 . A method of  claim 22  wherein the DNA sequences of the wild type DNA and the mutant DNA are known.  
     
     
         28 . A method of  claim 21  wherein the mutant DNA differs from wild type DNA by at least one base pair.  
     
     
         29 . A method of  claim 21  wherein the retention time used in the separation of the homoduplex or heteroduplex fraction was previously determined from a reference standard.  
     
     
         30 . A method of  claim 29  wherein the reference standard was obtained by separating a standard mixture of homoduplex and heteroduplex, having the same base pair sequence as the sample, by Matched Ion Polynucleotide Chromatography.  
     
     
         31 . An ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprising: a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein, the separation media having nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media.  
     
     
         32 . An ambient or low pressure device of  claim 31  wherein the separation media is a member selected from the group consisting of beads, capillary channels and monolith structure.  
     
     
         33 . An ambient or low pressure device of  claim 32  wherein the fixed unit of separation media comprise a fixed bed of separation media particles.  
     
     
         34 . An ambient or low pressure device of  claim 33  wherein the separation media particles are selected from the group consisting of organic polymer and inorganic particles having a nonpolar surface.  
     
     
         35 . An ambient or low pressure device of  claim 31  wherein the lower chamber is closed.  
     
     
         36 . An ambient or low pressure device of  claim 31  wherein the lower chamber has an open bottom portion.  
     
     
         37 . An ambient or low pressure device of  claim 36  in combination with a eluant container shaped to receive said lower chamber.  
     
     
         38 . An ambient or low pressure device of  claim 37  wherein the eluant chamber is a centrifuge vial.  
     
     
         39 . An ambient or low pressure device of  claim 37  wherein the cylinder is a member of an array of cylinders and the eluant container is a member of an array of eluant containers, and the array of cylinders and array of containers have matching configurations.  
     
     
         40 . An ambient or lower pressure separation system comprising a combination of multicavity separation plate having outer sealing edges, a multiwell collection plate and a vacuum system having a separation plate sealing means forming a sealed engagement with the outer sealing edges of the multicavity separation plate and a vacuum cavity receiving the multiwell collection plate; the multicavity separation plate including an array of tubes, each tube having an upper solution input chamber, a lower eluant receiving chamber with an bottom opening therein, and a fixed unit of separation media supported therein, the separation media having nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media; the multiwell collection plate having collection wells which are positioned to receive liquid from the bottom opening of the lower eluant receiving chamber.  
     
     
         41 . An ambient or lower pressure separation system of  claim 40  wherein the separation media is a member selected from the group consisting of beads, capillary channels and monolith structures.  
     
     
         42 . An ambient or low pressure device of  claim 41  wherein the fixed unit of separation media comprise a fixed bed of separation media particles.  
     
     
         43 . An ambient or low pressure device of  claim 42  wherein the separation media particles are selected from the group consisting of organic polymer and inorganic particles having a nonpolar surface.  
     
     
         44 . A method for removing a target DNA fragments having a predetermined base-pair length from a mixture of DNA fragments with an ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprising: a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein, the separation media having nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media comprising, the method comprising the steps of 
 a) applying a mixture of DNA fragments which may contain the target DNA fragments to a separation column containing media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent; and    b) removing the target DNA fragments from the media by contacting it with a second solvent solution containing a counterion and a concentration of driving solvent in cosolvent which has been predetermined to remove DNA fragments having the target DNA fragment base pair length from the media.