Plant Molecular Biology Reporter

, Volume 25, Issue 1–2, pp 1–9 | Cite as

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

  • Muriel Gaudet
  • Anna-Giulia Fara
  • Maurizio Sabatti
  • Elena Kuzminsky
  • Giuseppe Scarascia Mugnozza
Article

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.

Keywords

Allele-specific PCR Destabilizing mismatch PAMSA SNP genotyping Tailed primers 

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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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Muriel Gaudet
    • 1
  • Anna-Giulia Fara
    • 1
  • Maurizio Sabatti
    • 1
  • Elena Kuzminsky
    • 1
  • Giuseppe Scarascia Mugnozza
    • 1
  1. 1.Department of Forest Environment and Resources DISAFRIUniversity of TusciaViterboItaly

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