Galactomannans as a sieving matrix in capillary electrophoresis.
Purification of galactomannans including guaran, tara gum, and locust bean gum is described as well as their use as a sieving matrix in DNA sequencing by capillary electrophoresis (CE). Three methods of galactomannan purification were developed and tested using guaran. The first method is based on hydrolysis of proteins using alkali treatment and precipitation of guaran with acetone. The second method uses ion-exchange resins QAE Sephadex A-25 and SP Sephadex C-25 together with acetone precipitation. The third method is similar to the second one, except that it uses ion-exchange resins based on polystyrene, Source 30Q and Source 30S. Capillary zone electrophoresis of acetonitrile extracts from guaran revealed 4-5 characteristic major peaks and several minor peaks. Guar gum from different suppliers differed in the content of proteins. In purified guaran, protein peaks were detectable only using a 300-fold concentrate of extract. The content of proteins in the guaran purified using the third method was 0.001% m/m as determined by CE. The weight average molecular mass of purified guaran can be as large as 2.2 x 10(6). The purified galactomannans were used as a sieving matrix in DNA sequencing by CE. M13 DNA was sequenced to read lengths of about 600 bases in less than 90 min. Separation efficiencies exceeded 1 million theoretical plates for DNA fragments shorter than about 600 bases.
Dolník
V
V
Molecular Dynamics, Sunnyvale, CA 94085, USA. vlad.dolnik@am.apbiotech.com
Gurske
W A
WA
Padua
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A
D000097:Acetonitriles; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000302:isolation & purification; D003911:Dextrans; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005685:Galactans / Q000737:chemistry; D005690:Galactose / Q000031:analogs & derivatives; D008351:Mannans / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification; D053149:Plant Gums; D011134:Polysaccharides / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods; D013010:Sonication
D000097:Acetonitriles; D003911:Dextrans; D005685:Galactans; D008351:Mannans; D009838:Oligodeoxyribonucleotides; D053149:Plant Gums; D011134:Polysaccharides; D011506:Proteins; C012990:galactomannan; C038612:tara gum; D004247:DNA; C025614:sephadex; C007894:guar gum; C017471:locust bean gum; D005690:Galactose; C032159:acetonitrile