Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.) |
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Authors: | Muriel Gaudet Anna-Giulia Fara Maurizio Sabatti Elena Kuzminsky Giuseppe Scarascia Mugnozza |
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Institution: | (1) Department of Forest Environment and Resources DISAFRI, University of Tuscia, Via S. Camillo de Lellis, Viterbo, 01100, Italy |
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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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Keywords: | Allele-specific PCR Destabilizing mismatch PAMSA SNP genotyping Tailed primers |
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