Abstract
We tested applicability of a new genotyping technique to detect a low abundance CD17 (A → T) mutation of β-globin gene. The technique utilized a combined gap ligase chain reaction (Gap-LCR) and quantitative PCR (qPCR) methods. One pair of Gap-LCR primers was modified by adding specific sequences to the 5′ end of the upstream and the 3′ end of the downstream primer which served as a combining sequence for qPCR. First, specific mutation is detected using Gap-LCR; then, ligation products are detected by qPCR. Our results show that the amount of LCR products is directly proportional to the amount of template DNA. We further demonstrate that this technique detects a low abundance mutant DNA with a mutant/normal allele ratio as low as 1:10000. This technique was applied to detect a paternally inherited CD17 mutation from 53 maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. In conclusion, by combining Gap-LCR and qPCR technology we successfully established a highly sensitive technique to detect low abundance point mutations. This technique can be applied to detect fetal DNA point mutation in maternal plasma.
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Acknowledgments
The authors acknowledge Bing Deng (Diagnostic Centre of Molecular Biology, the Affiliated Children’s Hospital, Chongqing Medical University) for kindly providing DNA samples. This study was supported by the National Natural Science Foundation of China (No. 30801247) and by the National Key Technology R&D Program (No. 2006BA105A05).
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P. Yi and H. Jiang contributed equally to this work.
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Yi, P., Jiang, H., Li, L. et al. A New Genotyping Method for Detecting Low Abundance Single Nucleotide Mutations Based on Gap Ligase Chain Reaction and Quantitative PCR Assay. Cell Biochem Biophys 62, 161–167 (2012). https://doi.org/10.1007/s12013-011-9277-2
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DOI: https://doi.org/10.1007/s12013-011-9277-2