Concentration and Cleanup of Nucleic Acid Samples

Abstract

Methods and devices are described for concentration and cleanup of samples containing bio-molecule analytes (e.g., polynucleotides, such as DNA, RNA, PNA). Various embodiments provide for pH-mediated sample concentration and cleanup of nucleic acid samples with channel devices (e.g., cross-T format, microchannel devices).

1 . A sample-manipulation method, comprising: 
 (a) providing a channel device, said device including (i) an injection channel and a separation channel, each channel having a first end and a second end, with said separation channel intersecting the injection channel at a region between the ends of the injection channel; (ii) a first reservoir disposed for fluid communication with one of said ends of said injection channel; (iii) a first separation medium held within said injection channel; and (iv) a second separation medium held within said separation channel; wherein said second separation medium differs from said first separation medium;    (b) introducing a sample, including a polynucleotide-analyte component and one or more contaminants, into said injection channel;    (c) introducing a pH-modulating composition into said injection channel;    (d) stacking said polynucleotide-analyte component at a stacking region of said device defined by the intersection of the channels, and locating said one or more contaminants of said sample at a region between the stacking region and said second end of said injection channel; and    (e) electrophoresing said polynucleotide-analyte component down at least a portion of said separation channel, with at least most of said one or more contaminants remaining in said injection channel.    
     
     
         2 . A sample-manipulation method, comprising: 
 (a) providing a channel device, said device including (i) an elongate injection channel and an elongate separation channel, each channel having a first end and a second end, with said separation channel intersecting the injection channel at a region between the ends of the injection channel, and (ii) a loading region disposed for fluid communication with said first end of said injection channel;    (b) placing a sample containing a polynucleotide analyte and one or more contaminants into said loading region;    (c) applying a first driving force sufficient to cause said sample to move from said loading region into the injection channel;    (d) placing a basic solution into said loading region;    (e) applying a second driving force sufficient (i) to cause said basic solution to move from said loading region into the injection channel, thereby causing the polynucleotide analyte to stack in the region of the intersection of the channels, and (ii) to cause at least a portion of the one or more contaminants to move to a region between the intersection of the channels and said second end of said injection channel; and    (f) applying a third driving force sufficient to cause at least a portion of the stacked DNA to move into and along at least a portion of the separation channel, leaving behind in the injection channel at least most of the contaminants.    
     
     
         3 . The method of  claim 2 , wherein said injection and separation channels are arranged in a cross-T format.  
     
     
         4 . The method of  claim 2 , wherein each of said first, second and third driving forces comprises an electric field.  
     
     
         5 . The method of  claim 2 , wherein said basic solution comprises NaOH.  
     
     
         6 . The method of  claim 2 , wherein said polynucleotide analyte comprises DNA.  
     
     
         7 . The method of  claim 2 , further comprising placing a separation medium in each of said injection and separation channels.  
     
     
         8 . The method of  claim 7 , wherein the separation medium placed in said injection channel differs from the separation medium placed in the injection channel.  
     
     
         9 . The method of  claim 8 , wherein the separation media differ in one or both of concentration and composition.  
     
     
         10 . A sample-manipulation method, comprising: 
 (a) providing a channel device, said device including an injection channel and a separation channel, each channel having a first end and a second end, with said separation channel intersecting the injection channel at a region between the ends of the injection channel;    (b) introducing a sample, including a polynucleotide-analyte component and one or more contaminants, into said injection channel;    (c) introducing a pH-modulating composition into said injection channel;    (d) stacking said polynucleotide-analyte component at a stacking region of said device defined by the intersection of the channels, and locating said one or more contaminants of said sample at a region between the stacking region and said second end of said injection channel;    (e) electrophoresing said polynucleotide-analyte component down at least a portion of said separation channel, with at least most of said one or more contaminants remaining in said injection channel.    
     
     
         11 . The method of  claim 10 , wherein said injection and separation channels are arranged in a cross-T format.  
     
     
         12 . The method of  claim 10 , wherein said pH-modulating composition comprises a basic solution.  
     
     
         13 . The method of  claim 12 , wherein said basic solution comprises NaOH.  
     
     
         14 . The method of  claim 10 , wherein said polynucleotide-analyte component comprises DNA.  
     
     
         15 . The method of  claim 10 , further comprising placing a separation medium in each of said injection and separation channels.  
     
     
         16 . The method of  claim 15 , wherein the separation medium placed in said injection channel differs from the separation medium placed in the injection channel.  
     
     
         17 . The method of  claim 16 , wherein the separation media differ in one or both of concentration and composition.  
     
     
         18 . The method of  claim 10 , wherein said one or more contaminants include one or more dyes.  
     
     
         19 . The method of  claim 10 , wherein said sample is a DNA sequencing sample.  
     
     
         20 . A sample-manipulation method, comprising: 
 (a) providing a channel device, said device including (i) a first channel and a second channel, each channel having a first end and a second end, with said second channel intersecting the first channel at a region between the ends of the first channel; (ii) a loading region disposed for fluid communication with one of said ends of said first channel; (iii) a separation medium held within said first and second channels;    (b) introducing a sample, including a polynucleotide-analyte component and one or more contaminants, into said first channel by way of said loading region;    (c) stacking said polynucleotide-analyte component at a stacking region of said device defined by the intersection of the channels, and locating said one or more contaminants of said sample at a region between the stacking region and said second end of said first channel;    (d) electrophoresing said polynucleotide-analyte component down at least a portion of said second channel, with at least most of said one or more contaminants remaining in said first channel; and    (e) detecting for said polynucleotide-analyte component.    
     
     
         21 .- 31 . (canceled)