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Theoretical and Applied Genetics

, Volume 130, Issue 3, pp 597–607 | Cite as

An innovative SNP genotyping method adapting to multiple platforms and throughputs

  • Y. M. Long
  • W. S. Chao
  • G. J. Ma
  • S. S. Xu
  • L. L. QiEmail author
Original Article

Abstract

Key message

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility.

Abstract

Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3′ regions to significantly increase the amplification specificity of the two alleles and tailed at 5′ ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion–deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Keywords

Priming Element Individual SNPs Size Separation Common Reverse Primer Amplification Yield 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We thank Drs. Yiqun Weng and Zengcui Zhang for critically reviewing this manuscript. This research was supported by the USDA-ARS National Sclerotinia Initiative, Grant No. 5442-21220-028-00D and the USDA-ARS CRIS Project No. 3060-21000-039-00D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The experiments were performed in compliance with the current laws of the USA.

Supplementary material

122_2016_2838_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 15 kb)
122_2016_2838_MOESM2_ESM.xlsx (53 kb)
Supplementary material 2 (XLSX 52 kb)
122_2016_2838_MOESM3_ESM.docx (45 kb)
Supplementary material 3 (DOCX 44 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2016

Authors and Affiliations

  • Y. M. Long
    • 1
  • W. S. Chao
    • 2
  • G. J. Ma
    • 3
  • S. S. Xu
    • 2
  • L. L. Qi
    • 2
    Email author
  1. 1.Department of Plant SciencesNorth Dakota State UniversityFargoUSA
  2. 2.USDA-Agricultural Research Service, Northern Crop Science LaboratoryFargoUSA
  3. 3.Department of Plant PathologyNorth Dakota State UniversityFargoUSA

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