Detection Of Telomerase Activity And Single Nucleotide Polymorphism By Rolling Circle Amplification

Genome is the carrier of genetic information for organisms. Genetic information can be passed on to the next generation and be expressed by gene duplication. Gene mutation is closely related to the genetic diseases, so detecting genetic mutation is significant to the current genomic research and clinical diagnosis. Combining rolling circle amplification reaction with magnetic separation, fluorescence and resonance light scattering techniques, we established new methods for the detection of telomerase activity and single nucleotide polymorphism (SNP).In this paper, we have established a new method to detect the telomerase activity based on Branched Rolling Circle Amplification (BRCA). Telomerase, as a kind of reverse transcriptase, can extend biotinylated telomere with repeat sequences. The extended products are magnetically separated using streptavidin (STV) coated magnetic beads based on the interaction between STV and biotin. The designed primers with 12 bases are perfectly complementary to the extension products. After hybridization between the primers and the extended products, the primers are magnetically separated again and then released into solution by heating. The primers can start the BRCA reaction. The BRCA reaction products are detected using fluorescence dye, SYBR Green I. With this method telomerase activity can be detected from 20,000 lung cancer cells.Combining BRCA with resonance scattering technology, we have proposed a new method for detection of SNP. Designed padlock probe perfectly matching with mutant DNA at both ends can be ligated and circularized using Ampligase. And the mutant DNA can directly prime BRCA reaction. Correspondingly, the wild DNA contains a mismatched base with the padlock probe. So the probe can't be circularized and the BRCA reaction can't take place, and only a limited linear amplification occurs. When the BRCA products are added to strong acid medium (HClO4), the nucleic acids are denatured and aggregated into big particles, producing enhanced resonance light scattering. With this method we can sensitively distinguish 1 pmol/L mutant and wild DNA, and accurately determine the mutant frequency as low as 1.0 %.