A versatile microfabricated platform for electrophoresis of double- and single-stranded DNA.
We demonstrate a versatile microfabricated electrophoresis platform, incorporating arrays of integrated on-chip electrodes, heaters, and temperature sensors. This design allows a range of different sieving gels to be used within the same device to perform separations involving both single- and double-stranded DNA over distances on the order of 1 cm. We use this device to compare linear and cross-linked polyacrylamide, agarose, and thermo-reversible Pluronic-F127 gels on the basis of gel casting ease, reusability, and overall separation performance using a 100 base pair double-stranded DNA ladder as a standard sample. While cross-linked polyacrylamide matrices provide consistently high-quality separations in our system over a wide range of DNA fragment sizes, Pluronic gels also offer compelling advantages in terms of the ability to remove and reload the gel. Agarose gels offer good separation performance, however, additional care must be exercised to ensure consistent gel properties as a consequence of the need for elevated gel loading temperatures. We also demonstrate the use of denaturing cross-linked polyacrylamide gels at concentrations up to 19% to separate single-stranded DNA fragments ranging in size from 18 to 400 bases in length. Primers differing by 4 bases at a read length of 30 bases can be separated with a resolution of 0.9-1.0 in under 20 min. This level of performance is sufficient to conduct a variety of genotyping assays including the rapid detection of single nucleotide polymorphisms (SNPs) in a microfabricated platform. The ability to use a single microelectrophoresis system to satisfy a wide range of separation applications offers molecular biologists an unprecedented level of flexibility in a portable and inexpensive format.
Ugaz
Victor M
VM
Department of Chemical Engineering, The University of Michigan, Ann Arbor, 48109-2136, USA.
Lin
Rongsheng
R
Srivastava
Nimisha
N
Burke
David T
DT
Burns
Mark A
MA
D000180:Acrylic Resins / Q000737:chemistry; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification*; D004566:Electrodes; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D020442:Poloxamer; D020641:Polymorphism, Single Nucleotide; D013696:Temperature
D000180:Acrylic Resins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; C016680:polyacrylamide gels; D020442:Poloxamer; D004247:DNA