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Advances and Clinical Practice in Pyrosequencing pp 3–12Cite as

Preparation of Single-Stranded DNA for Pyrosequencing by LATE-PCR

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Abstract

To establish a simple method for preparing single-stranded DNA templates for pyrosequencing, the Linear-after-the-exponential (LATE)-PCR technology on the basis of Taq DNA polymerase without hot-start capacity was applied to amplify a 78-bp sequence (containing the SNP6 site), and the PCR-enhancing reagents (glycerol and BSA) were used to increase the efficiency and specialization, much more before the reagents A and B were designed to eliminate the impurity (limited primers, PPi, dNTPs, and so on), and 1–2 μL LATE-PCR products with simply treatment can be used in pyrosequencing directly. Then, five SNPs related with human breast-cancers in the BRCA1 gene were investigated, and the programs had no nonspecific signals that were made of theoretic sequences. Moreover, the genotyping of the SNPs could also be distinguished easily. The results indicated that this method can be used to prepare high quality single-stranded DNA templates for pyrosequencing and allows pyrosequencing be lower in cost, simpler in operation, and easier in automation, and the cross-contamination from sample preparation was also reduced.

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Author information

Authors and Affiliations

  1. China Pharmaceutical University, Nanjing, China

    Huiyong Yang

  2. Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing, China

    Zhiyao Chen, Bingjie Zou & Guohua Zhou

  3. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu, 210009, China

    Qinxin Song

  4. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, China

    Chao Liang

Authors
  1. Huiyong Yang
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  2. Chao Liang
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  3. Zhiyao Chen
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  4. Bingjie Zou
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  5. Qinxin Song
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  6. Guohua Zhou
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Corresponding authors

Correspondence to Qinxin Song or Guohua Zhou .

Editor information

Editors and Affiliations

  1. State Key Laboratory of Analytical Chemi, Medical School of Nanjing University, Nanjing, Jiangsu, China

    Guohua Zhou

  2. Key Laboratory of Drug Quality Control, China Pharmaceutical University, Nanjing, Jiangsu, China

    Qinxin Song

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Yang, H., Liang, C., Chen, Z., Zou, B., Song, Q., Zhou, G. (2016). Preparation of Single-Stranded DNA for Pyrosequencing by LATE-PCR. In: Zhou, G., Song, Q. (eds) Advances and Clinical Practice in Pyrosequencing. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3308-2_1

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  • DOI: https://doi.org/10.1007/978-1-4939-3308-2_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3306-8

  • Online ISBN: 978-1-4939-3308-2

  • eBook Packages: Springer Protocols

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