High-Throughput, High-Sensitivity Genetic Mutation Detection by Tandem Single-Strand Conformation Polymorphism/Heteroduplex Analysis Capillary Array Electrophoresis
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- 作者：Igor V. Kourkine ; Christa N. Hestekin ; Brett A. Buchholz ; and Annelise E. Barron
- 刊名：Analytical Chemistry
- 出版年：2002
- 出版时间：June 1, 2002
- 年：2002
- 卷：74
- 期：11
- 页码：2565 - 2572
- 全文大小：242K
- 年卷期：v.74,no.11(June 1, 2002)
- ISSN：1520-6882
文摘
We present the first optimization of linear polyacrylamide(LPA)-based DNA separation matrixes for an automatedtandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method,implemented in capillary arrays dynamically coated withpoly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed bothSSCP and HA species to be produced in one step in asingle tube and distinguished in a single electrophoreticanalysis. A simple, two-color fluorescent sample labelingand detection strategy enabled unambiguous identificationof all DNA species in the electropherogram, both single-and double-stranded. Using these protocols and a panelof 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 polyDuramidewall coating was found to be essential for resolution ofCAE-SSCP/HA peaks and yielded sensitive mutationdetection in all 11 p53 samples initially studied. A largerset of 32 mutant DNA specimens was then analyzed usingthese optimized tandem CAE-SSCP/HA protocols andmaterials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lowersensitivity on its own (93% for SSCP and 75% for HA).This simple, highly sensitive tandem SSCP/HA mutationdetection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to theease of the capillary coating protocol and the low viscosityof the matrix.