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Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.)

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Abstract

The wide development of single nucleotide polymorphism (SNP) markers also in non-model species increases the need for inexpensive methods that do not require sophisticated equipment and time for optimization. This work presents a new method for polymerase chain reaction (PCR) amplification of multiple specific alleles (PAMSA), which allows efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. This improved PAMSA requires only three unlabeled primers: a common reverse primer and two allele-specific primers having a tail of different length to differentiate the two SNP alleles by the size of amplification products on agarose gel. A destabilizing mismatch within the five bases of the 3′ end is also added to improve the allele specificity. To validate the accuracy of this method, 94 full-sib individuals were genotyped with three SNPs and compared to the genotypes obtained by cleaved amplified polymorphic sequence (CAPS) or derived CAPS. This method is flexible, inexpensive, and well suited for high throughput and automated genotyping.

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Abbreviations

ARMS:

amplification refractory mutation system

AS-PCR:

allele-specific polymerase chain reaction

Bi-PASA:

bidirectional-PASA

CAPS:

cleaved amplification polymorphic site

dCAPS:

derived cleaved amplification polymorphic site

PAMSA:

PCR amplification of multiple specific alleles

PASA:

PCR allele-specific amplification

SNP:

single nucleotide polymorphisms

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Acknowledgments

The authors thank Véronique Jorge and Claudia Mattioni for their critical reading of the manuscript, Dermot Browne for correction of English writing, and Isacco Beritognolo for his technical advice and assistance. This work was supported by grants from the European Project QLK5-CT-2002-00953 (POPYOMICS) and the Italian project MIUR PRIN 2005072892_001.

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Authors and Affiliations

  1. Department of Forest Environment and Resources DISAFRI, University of Tuscia, Via S. Camillo de Lellis, Viterbo, 01100, Italy

    Muriel Gaudet, Anna-Giulia Fara, Maurizio Sabatti, Elena Kuzminsky & Giuseppe Scarascia Mugnozza

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  1. Muriel Gaudet
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  2. Anna-Giulia Fara
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  3. Maurizio Sabatti
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  4. Elena Kuzminsky
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  5. Giuseppe Scarascia Mugnozza
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Correspondence to Muriel Gaudet.

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Gaudet, M., Fara, AG., Sabatti, M. et al. Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.). Plant Mol Biol Rep 25, 1–9 (2007). https://doi.org/10.1007/s11105-007-0003-6

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  • DOI: https://doi.org/10.1007/s11105-007-0003-6

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