Multiplexed p53 Mutation Detection by Free-Solution Conjugate Microchannel Electrophoresis with Polyamide Drag-Tags

Abstract

We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide "drag-tags" was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reaction products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reaction was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free-solution conjugate electrophoresis (FSCE) in both capillaries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring.

A 265-Base DNA Sequencing Read by Capillary Electrophoresis with No Separation Matrix

“…33 A similar decrease is expected for FSCE sequencing on microchips; sequencing of 265 bases is likely to be achieved in ~3 minutes. The largest protein drag-tags (54 and 72 repeats of the monomer amino acid block) showed significant monodispersity and the 72mer is predicted to have the friction (α = 152) necessary to sequence at least 400 bases and through an entire exon for medical diagnostics.…”

“…24,33 In short, the protein is mixed with a 10:1 molar excess of sulfo-SMCC, vortexed for 1 hour, and lyophilized after using a CentriSep gel filtration column (Princeton Separations, Adelphia, NJ) to remove excess sulfo-SMCC. The thiol-terminated ssDNA oligomer is reduced with a 20:1 molar excess of TCEP (tris(2-carboxyethylphosphine), Thermo) at 40 °C for 100 min, desalted and separated from excess TCEP with a CentriSep column and then incubated with a 100:1 molar excess of the activated drag-tag at room temperature for 4-18 h.…”

High resolution DNA separations using microchip electrophoresis

“…Moreover, the total development time of reactions is considerably short compared to most typing methods in terms of those requiring thermal cycling [42]. SBE reactions entail annealing a primer one base removed from the locus containing the SNP to a particular DNA template.…”

“…Recently, the Barron group demonstrated the microchip-based separation of multiplexed reactions to probe 16 p53 loci with 96% accuracy using ELFSE and microchip CE. With only a denaturing buffer containing a EOF suppressant as an electrophoretic medium, extension units harboring 16 unique, monodispersed, uncharged polyamide drag-tags facilitated the size dependent separation of species within 70 s using a commercial glass microchip [42].…”

Further improvement of hydrostatic pressure sample injection for microchip electrophoresis

“…[4,5]. The use of microfabricated devices for DNA sizing recently has become trends because these devices greatly reduce sample volume, separation time and cost per analysis [6][7][8][9][10][11][12]. Addition of viscous polymers into the separation buffer generally is necessary for DNA sizing [13,14], which considerably increases the resolving ability of electrophoresis.…”