We present the first optimization of linear polyacrylamide (LPA)-based DNA separation matrixes for an automated tandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method, implemented in capillary arrays dynamically coated with poly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed both SSCP and HA species to be produced in one step in a single tube and distinguished in a single electrophoretic analysis. A simple, two-color fluorescent sample labeling and detection strategy enabled unambiguous identification of all DNA species in the electropherogram, both single- and double-stranded. Using these protocols and a panel of 11 p53 mutant DNA samples in comparison with wild-type, we employed high-throughput capillary array electrophoresis (CAE) to carry out a systematic and simultaneous optimization of LPA weight-average molar mass (Mw) and concentration for SSCP/HA peak separation. The combination of the optimized LPA matrix (6% LPA, Mw 600 kDa) and a hydrophilic, adsorbed polyDuramide wall coating was found to be essential for resolution of CAE-SSCP/HA peaks and yielded sensitive mutation detection in all 11 p53 samples initially studied. A larger set of 32 mutant DNA specimens was then analyzed using these optimized tandem CAE-SSCP/HA protocols and materials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA). This simple, highly sensitive tandem SSCP/HA mutation detection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to the ease of the capillary coating protocol and the low viscosity of the matrix.
Physically adsorbed (dynamic) polymeric wall coatings for microchannel electrophoresis have distinct advantages over covalently linked coatings. In order to determine the critical factors that control the formation of dynamic wall coatings, we have created a set of model polymers and copolymers based on N,N-dimethylacrylamide (DMA) and N,N-diethylacrylamide (DEA), and studied their adsorption behavior from aqueous solution as well as their performance for microchannel electrophoresis of DNA. This study is revealing in terms of the polymer properties that help create an "ideal" wall coating. Our measurements indicate that the chemical nature of the coating polymer strongly impacts its electroosmotic flow (EOF) suppression capabilities. Additionally, we find that a critical polymer chain length is required for polymers of this type to perform effectively as microchannel wall coatings. The effective mobilities of double-stranded (dsDNA) fragments within dynamically coated capillaries were determined in order to correlate polymer hydrophobicity with separation performance. Even for dsDNA, which is not expected to be a strongly adsorbing analyte, wall coating hydrophobicity has a deleterious influence on separation performance.
The acrylonitrile complexes Pt(diphos)(CH 2 CHCN) (diphos ) dppe (1), dcpe (2); dppe ) Ph 2 PCH 2 CH 2 PPh 2 , dcpe ) Cy 2 PCH 2 CH 2 PCy 2 , Cy ) cyclo-C 6 H 11 ) are catalyst precursors and, for some substrates, resting states, during addition of P-H bonds in primary and secondary phosphines across the CdC double bond of acrylonitrile (hydrophosphination). Oxidative addition of P-H bonds to related catalyst precursors gives the phosphido hydride complexes Pt(diphosAcrylonitrile does not insert into the Pt-H bond of these hydrides to give cyanoethyl ligands; the putative products, the phosphido complexes Pt(diphos 11)) were prepared independently and found to be stable to P-C reductive elimination. Instead, catalysis appears to occur by selective insertion of acrylonitrile into the Pt-P bond to yield the alkyl hydrides Pt(diphos)[CH(CN)CH 2 PRR′](H), followed by C-H reductive elimination and regeneration of 1 or 2. This insertion was observed directly in model methyl phosphido complexes M(dppe)-(Me)(PRR′) (M ) Pt, R ) H, R′ ) Mes* (12), R ) R′ ) Mes (13); M ) Pd, R ) H, R′ ) Mes* (17)), yielding M(dppe)[CH(CN)CH 2 PRR′](Me), (14, 15, 18). Similarly, treatment of Pt(dcpe)-(PHMes*)(H) (22) with acrylonitrile gives Pt(dcpe)[CH(CN)CH 2 PHMes*](H) (24) as a mixture of diastereomers; the isomeric Pt(dcpe)[PMes*(CH 2 CH 2 CN)](H) (25), which was prepared independently, was also observed during this reaction. Both 24 and 25 decompose in the presence of acrylonitrile to form Pt(dcpe)(CH 2 CHCN) (2) and PHMes*(CH 2 CH 2 CN) (3a). The C-H reductive elimination step was modeled by studies of Pt(dcpe)[CH(Me)(CN)](H) (26). Another isomer, Pt(dcpe)[CH(Me)(CN)](PHMes*) (29), which formally results from insertion of acrylonitrile into the Pt-H bond of 22, was formed by decomposition of complex 2 during catalysis. Complex 29 is inactive in catalysis but decomposes to partially regenerate the active catalyst 2. The cyanoethyl compounds Pt(dcpe)(CH 2 CH 2 CN)(PHMes*) (11), trans-Pt-(PPh 3 ) 2 (CH 2 CH 2 CN)(Br), and PMes 2 (CH 2 CH 2 CN) (23) were structurally characterized by X-ray crystallography.
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