High-throughput single molecule screening and selective single molecule PCR for early stage disease diagnosis based on capillary electrophoresis

The purpose of this research was to develop high throughput approaches for single molecule and single cell screening for early stage disease diagnosis. High sensitivity as well as high selectivity is essential for the success screening.; We first demonstrated a laser-induced fluorescence imaging method that allows screening many single molecules at a time based on their electrophoretic mobilities. YoYo-I labeled DNA molecules were tracked by a charge-coupled device (CCD) camera under a microscope while migrating in the electric field. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. Each measurement only requires a few milliseconds to complete, which opens up the possibility of screening tens of thousands of molecules every second. The results correlate well with normal capillary electrophoresis (CE) experiments for the same samples under identical separation conditions.; We also developed on-line capillary polymerase chain reaction (PCR) coupled with laser-induced fluorescence detection for individual DNA molecules. A single 30-μm-i.d. fused-silica capillary was used both as the reaction vessel and for isolating single molecules. Because of the small inside diameter of the capillary, PCR-amplified DNA fragments from single molecules were localized in the capillary, providing discrete product zones with concentrations at readily detectable levels. By counting the number of peaks in the capillary via electromigration past a detection window, the number of starting DNA molecules could be determined. Amplification of the 110-bp fragment from an individual human β-globin gene and the 142-bp fragment from an individual HIV-1 DNA was demonstrated.; Finally, we achieved the direct online capillary PCR amplification from crude biological samples. Human β-actin gene within individual lymphoblast cells was successfully amplified and detected in the capillary with no extra sample preparation step. This would provide highly selective and sensitive disease diagnosis at a very early stage when there are only a few infected cells. The problem reduces to identifying the suitable primer pairs for each disease marker. The feasibility for continuous-flow PCR monitoring, along with the well-developed capillary array electrophoresis techniques, will provide the high throughput and high sensitivity for large-scale clinical diagnosis.