Highly Sensitive Mutation Detection Based on Single-Molecular PCR and Hydrogel-immobilized Bead-chip
We have developed a novel method for mutation detection based on microbeads from solid-phase single-molecule PCR in a fine water-phase compartment formed in water-in-oil (w/o) emulsions, coupling with a new detection platform: Hydrogel-immobilized Bead-chip. In this method, DNA fragments were distributed over the emulsion as templates, where beads crosslinked with primers and other PCR components were encapsulated to form multiple reaction compartments. One compartment only has one template molecule and one bead, or only one of them, or none. After the recovery of beads and the preparation of ss-DNA, 3-D polyacrylamide gel network was used as a carrier to immobilize the beads on the glass chip. To decode the beads, two allele-specific fluorescence probes, the sequences of which only have one-base difference, were annealed to the amplicons on beads surface. A perfect discrimination of single-base mismatch was achieved by treating the beaded chip with electrophoresis. The proportion of mutant is determined by counting the numbers of the beads with different color on the chip. We have successfully detected the mutants with the ratio of 1:100 and 1:1000 (mutant:wild-type) by counting the red and green beads immobilized on the chip. Therefore, this system is a powerful tool for analyzing a small amount of mutant in a sample, and opens a new way for early cancer diagnosis.
Single-Molecular PCR Hydrogel Immobilized Beaded-Chip Mutation Detection OLR-1 gene
Huan Huang Guohua Zhou
Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002 P.R.China; School of Life S Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002 P.R.China; Medical School,
国际会议
The 5th International Forum on Post-genome Technologies(5IFPT)(第五届国际后基因组生命科学技术学术论坛)
苏州
英文
2007-09-10（万方平台首次上网日期，不代表论文的发表时间）