Analytical and Bioanalytical Chemistry

, Volume 408, Issue 12, pp 3113–3123 | Cite as

Pyrosequencing with di-base addition for single nucleotide polymorphism genotyping

  • Dan Pu
  • Chengguang Mao
  • Lunbiao Cui
  • Zhiyang Shi
  • Pengfeng XiaoEmail author
Research Paper


We develop color code-based pyrosequencing with di-base addition for analysis of single nucleotide polymorphisms (SNPs). When a di-base is added into the polymerization, one or several two-color code(s) containing the type and the number of incorporated nucleotides will be produced. The code information obtained in a single run is useful to genotype SNPs as each allelic variant will give a specific pattern compared to the two other variants. Special care has to be taken while designing the di-base dispensation order. Here, we present a detailed protocol for establishing sequence-specific di-base addition to avoid nonsynchronous extension at the SNP sites. By using this technology, as few as 50 copies of DNA templates were accurately sequenced. Higher signals were produced and thus a relatively lower sample amount was required. Furthermore, the read length of per flow was increased, making simultaneous identification of multiple SNPs in a single sequencing run possible. Validation of the method was performed by using templates with two SNPs covering 37 bp and with three SNPs covering 58 bp as well as 82 bp. These SNPs were successfully genotyped by using only a sequencing primer in a single PCR/sequencing run. Our results demonstrated that this technology could be potentially developed into a powerful methodology to accurately determine SNPs so as to diagnose clinical settings.

Graphical Abstract

Conventional pyrosequencing adds one base (A, G, C, or T) at a time to determine the SNP site (left). Pyrosequencing with di-base addition adds di-base AG, AC, AT, CT, GC or GT at a time to determine the SNP site (right). Higher signals at SNP site will be produced due to the addition of di-bases


Pyrosequencing Single nucleotide polymorphisms (SNPs) Di-base Color code 



The work was funded by the Major State Basic Research Development Program of China (2012CB517706) and the National Natural Science Foundation of China (61571114).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2016_9359_MOESM1_ESM.pdf (931 kb)
ESM 1 (PDF 931 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Dan Pu
    • 1
  • Chengguang Mao
    • 1
  • Lunbiao Cui
    • 2
  • Zhiyang Shi
    • 2
  • Pengfeng Xiao
    • 1
    Email author
  1. 1.State Key Laboratory of Bioelectronics, School of Biological Science and Medical EngineeringSoutheast UniversityNanjingChina
  2. 2.Key Laboratory of Enteric Pathogenic Microbiology (NHFPC)Jiangsu Provincial Center for Disease Control and PreventionNanjingChina

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