An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications.
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.
de Arruda
Maurício Papa
MP
Laboratório de Chiroptera/Departamento de Zoologia e Botânica, Universidade Estadual Paulista (UNESP), Campus S.J. Rio Preto, São Paulo, Brazil. arrudabio@gmail.com
Gonçalves
Evonnildo Costa
EC
Schneider
Maria Paula Cruz
MP
da Silva
Artur Luiz da Costa
AL
Morielle-Versute
Eliana
E
D000818:Animals; D001001:Anura / Q000235:genetics; D016477:Artifacts; D001483:Base Sequence; D004396:Coloring Agents / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism*; D005838:Genotype; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling; D013696:Temperature
D004396:Coloring Agents; D017931:DNA Primers