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Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution

Nature Protocols volume 4, pages 984–991 (2009)Cite this article

Abstract

This protocol describes a simple, convenient and sensitive single-nucleotide polymorphism (SNP) genotyping method using an optically amplifying cationic conjugated polymer and single base primer extension reaction. The fluorescence resonance energy transfer (FRET) efficiency between the conjugated polymer (PFP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide}) and a fluorescein-labeled dNTP (dNTP-Fl) is correlated to the incorporation of the dNTP-Fl into an allele-specific primer; incorporation occurs by a single base extension reaction when the target DNA and the primer are complementary at the SNP site. By triggering the FRET from PFP to fluorescein and measuring the change in fluorescence intensity of samples, the SNP genotypes can be discriminated. In comparison with other SNP genotyping methods, this protocol simplifies procedures and improves sensitivity by eliminating the need for primer labeling, cumbersome workups, chemical/enzymatic coupling reactions and sophisticated instruments. The assay takes about 2 h for PCR amplification followed by 5.5–7.5 h to obtain the genotypes.

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Figure 1: Outline of CP-based SNP genotyping.
Figure 2: Fluorescence assay results for single-nucleotide polymorphism (SNP) site (rs2241715: G>T) using CP-based SNP genotyping method.
Figure 3: Genotyping results for single-nucleotide polymorphism (SNP) site (rs1800469: C>T) using CP-based SNP genotyping method.
Figure 4: Genotyping results for single-nucleotide polymorphism (SNP) site (rs2241715:G>T) using CP-based SNP genotyping method.

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Acknowledgements

We thank all the individuals tested. This work was supported in part by grants from the “100 Talents” program of the Chinese Academy of Sciences, the National Natural Science Foundation of China (nos. 20725308, 30621063 and 30872929), the National High-Tech R&D Program of China (nos. 2006AA02Z130 and 2006AA02A412), the Major Research Plan of China (no. 2006CB932102) and the Key Project for the Infectious Diseases of China (no. 2008ZX10002-016).

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Author notes

  1. Xinrui Duan and Wei Yue: These authors contributed equally to this work.

Authors and Affiliations

  1. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China

    Xinrui Duan, Libing Liu, Yuliang Li, Daoben Zhu & Shu Wang

  2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China

    Wei Yue, Fuchu He & Gangqiao Zhou

  3. College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei Province, China

    Zhengping Li

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Corresponding authors

Correspondence to Gangqiao Zhou or Shu Wang.

Supplementary information

Supplementary Data 1

The sequencing results of 26 samples at SNP site rs1800469. (PDF 437 kb)

Supplementary Data 2

The sequencing results of 50 samples at SNP site rs2241715. (PDF 912 kb)

Supplementary Fig. 1

Genotyping results for SNP site (rs1800469: C<T) using CP–based SNP genotyping method with sample number as symbol. FRET ratio of PFP to fluorescein (I530nm/I425nm) using C allele primer as X axis, and that using T allele primer as Y axis. No-template control (NTC) is used as a blank. (PDF 58 kb)

Supplementary Fig. 2

Genotyping results for SNP site (rs2241715:G<T) using CP–based SNP genotyping method with sample number as symbol. FRET ratio of PFP to fluorescein (I530nm/I425nm) using G allele primer as X axis, and that using T allele primer as Y axis. No-template control (NTC) is used as a blank. (PDF 60 kb)

Supplementary Table 1

The purpose and sequences of primers for PCR of SNPs rs1800469 and rs2241715. (PDF 47 kb)

Supplementary Table 2

Purpose and sequences of primers for single base extension reaction of SNPs rs1800469 and rs2241715. (PDF 46 kb)

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Duan, X., Yue, W., Liu, L. et al. Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution. Nat Protoc 4, 984–991 (2009). https://doi.org/10.1038/nprot.2009.70

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