An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications
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Data
2010-04-01
Autores
de Arruda, Mauricio Papa [UNESP]
Goncalves, Evonnildo Costa
Cruz Schneider, Maria Paula
da Costa da Silva, Artur Luiz
Morielle-Versute, Eliana [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Springer
Resumo
We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5' end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.
Descrição
Palavras-chave
Genotyping, Labeled primer, Microsatellites, Tailed primer, Unspecific amplifications, Laser detection systems
Como citar
Molecular Biology Reports. Dordrecht: Springer, v. 37, n. 4, p. 2031-2036, 2010.