Abstract for presentation at 11th International Congress of Human Genetics

In situ detection of small target sequences and SNP genotyping by rolling circle amplification of padlock probe

  • Jee Kyhm, Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, Korea, Korea
  • Sung Shim, Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, Korea, Korea
  • Min Sung, Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, Korea, Korea
  • Chul Lee, Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, Korea, Korea
  • Youl Cho, Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, Korea, Korea

    • Padlock probes (PLP), so called CLiP (circularizable ligation probes) or OPC (open circle probe), have high specificity and excellent selectivity not only to detect small gene sequence but also to discriminate gene sequences with single base difference. In this study, padlock probe were synthesized about 120 nucleotide length by PCR method instead of chemical synthesis. Padlock probe consists of 5’ and 3’ target complementary region and a non-human DNA linker. The 5’ and 3’ terminal regions of probes, respectively 20 and 12~20 nucleotides, were hybridized to a target, the nicked circles can be sealed by Tth DNA ligase. The only circles bound to the target were amplified to visualize through the rolling circle amplification by Phi 29 DNA polymerase and the product was detected by green- or red-labeled detection probes.
    A padlock probe for exon 3 of factor IX was generated by PCR method. This probe hybridized successfully to the target region as small as 40bp in interphase nuclei. Two signals in female and one signal in male interphase nuclei were detected.
    We applied this method to in situ genotyping of C677T polymorphic site in MTHFR gene. Two probes were hybridized differentially to the target in interphase based on sequence variation. Two green (CC) or two red (TT) signals were detected in homozygote and one green and one red signal were detected in heterozygote. These probes can be used in real-time PCR detection system.
    In conclusion, this combined technique of padlock probes and rolling circle amplification are very useful to detect short sequence as well as SNP.

    Conference Organiser - ICMS Pty Ltd