Single-reaction for SNP genotyping on agarose gel by allele-specific PCR in black poplar (Populus nigra L.)

被引：34

作者：

Gaudet, Muriel ^{[1
]}

Fara, Anna-Giulia ^{[1
]}

Sabatti, Maurizio ^{[1
]}

Kuztninsky, Elena ^{[1
]}

Mugnozza, Giuseppe Scarascia ^{[1
]}

机构：

[1] Univ Tuscia, Dept Forest Environm & Resources, DISAFRI, I-01100 Viterbo, Italy

来源：

PLANT MOLECULAR BIOLOGY REPORTER | 2007年 / 25卷 / 1-2期

关键词：

allele-specific PCR; destabilizing mismatch; PAMSA; SNP genotyping; tailed primers;

D O I：

10.1007/s11105-007-0003-6

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：1 / 9

页数：9

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