Development of Multiplex Quantitative Genetic Analysis Technology Using Capillary Electrophoresis-Based Single Strand Conformation Polymorphism

Abstract

For accurate genetic analysis, sequence-based detection of genetic materials should be performed quantitatively. Sequencing analysis, the “gold standard” of sequence-based genetic analysis has limitations on its qualitative nature and relatively high cost. DNA/DNA hybridization-based genetic tools can be used as alternative sequence-based detection method, but mismatched hybridization is inevitable so that the methods cannot guarantee accuracy of the analysis. Electrophoresis-based technologies, on the other hand, can be more useful because they analyze genetic molecules in a hybridization-free manner. Among various electrophoresis-based methods, capillary electrophoresis (CE)-based single strand conformation polymorphism (SSCP) is one of the most commonly used methods since it is a versatile and inexpensive technique. CE-SSCP has been mainly used for mutation detection method, but it has much potential as a multiplex and quantitative genetic analysis method since the method can sequence-specifically separate DNA molecules and quantitative information is also obtainable.CE-SSCP, which separates DNA molecules by conformational difference of ssDNA and quantifies them by fluorescence intensity, can detect multiple DNA or RNA sequences quantitatively. To develop multiplex analysis methods appropriate for various genetic analyses, such as pathogen gene marker detection, expression analysis, and DNA copy number measurement, here we introduced various strategies to improve resolution, dynamic range, and precision of the analysis.First, to improve the resolution of the CE-SSCP analysis, strategies using species specific primer and a novel separation medium were introduced. Pathogen gene marker detection was used as a model for the researches on resolution improvement because the markers were highly homologous. Especially, the strategy with a PEO-PPO-PEO triblock copolymer solution as a new sieving medium was extremely effective, and now more than ten targets can be analyzed with sufficient resolution. In addition, this system has been translated into a microchip device format.Second, to improve the dynamic range of CE-SSCP-based measurement, amplification of sequence-tagged templates or probes using common primer sets in unequal amount was suggested and be proven its effectiveness in a analysis requiring a wide dynamic range, such as expression analysis. In particular, stuffer-free multiplex ligation-dependent probe amplification (MLPA) and the high-resolution CE-SSCP was used for various expression analysis. By the approaches, the dynamic range was greatly improved, and over a hundred fold change could be analyzed precisely.Lastly, the precision of CE-SSCP-based measurement was improved to be good enough for discrimination of single copy difference. To accomplish this, we first suggested a strategy using real-time PCR-based maximum doubling cycle (CMD) determination, which can be applied for any end-point PCR analysis. Also, the stuffer-eliminated short probes were very effective for a precise determination of DNA copy number, and the long probe length of conventional MLPA has been indicated as a main cause of its semi-quantitative nature.