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Improved PCR-BSP Assay for Multiplex Methylation Pattern Analysis in Minimal Amount of DNA

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Abstract

Cell-specific DNA methylation pattern detection is of great importance for the tumorigenesis and differentiation studies. Comparatively, large amounts of DNA were needed for traditional methods of DNA methylation pattern detection, and therefore, more sensitive method for high throughput analysis with a limited amount of DNA is needed. With Mouse 3T3 cells, we developed new multiplex-nested PCR technologies for bisulfite-assisted genomic sequencing PCR (BSP) methylation pattern detection method. Primers step add-in method and templates precipitation methods efficiently increase the throughput of the assay, and the nested PCR method also increased the sensitivity. The optimized assay could successfully detect 15 sequences of methylation pattern with a minimal amount of DNA (500–1,000 cells of genome DNA).

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Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (no. 30771191), and the Key Project of Science & Technology Commission of Shanghai Municipality (no. 071409004).

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Authors and Affiliations

  1. Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Jianhui Wang & Minghui Yu

  2. College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China

    Kai Li, Junhua Xiao & Yuxun Zhou

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  1. Jianhui Wang
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  2. Minghui Yu
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  3. Kai Li
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  4. Junhua Xiao
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  5. Yuxun Zhou
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Correspondence to Yuxun Zhou.

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Wang, J., Yu, M., Li, K. et al. Improved PCR-BSP Assay for Multiplex Methylation Pattern Analysis in Minimal Amount of DNA. Mol Biotechnol 42, 333–340 (2009). https://doi.org/10.1007/s12033-009-9169-5

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  • DOI: https://doi.org/10.1007/s12033-009-9169-5

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