An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

Abstract

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.

Whole-genome genotyping of grape using a panel of microsatellite multiplex PCRs

“…This kind of techniques would offer an inexpensive alternative to the commercial synthesizing of custom labeled primers for multiplex genotyping. However, it should be noted that additional optimization steps may be needed because sometimes the three primers approach seem to decrease PCR efficiency (de Arruda et al 2010). …”

Transferability of microsatellites for studies on the social behavior of the tufted capuchin monkey (genus Sapajus)

“…We labeled the forward primer of each locus with an M13 tail (Schuelke, 2000) and added a fluorescently labeled universal M13 primer to the polymerase chain reaction (PCR) after the final extension step, as suggested by de Arruda et al (2010). Our 10-µL reaction volume contained 2 µL DNA, 5 µL GoTaq Colorless Master Mix (Promega, USA), 1 µL bovine serum albumin (Fermentas, Lithuania), 1 µL forward primer, and 1 µL reverse primer.…”

Universal tail sequence-SSR applied to molecular characterization of tropical maize hybrids

“…The inclusion of an extended fluorescently labeled tail in the SSR primers has been proposed to improve automation and reduce genotyping costs (Cryer et al, 2005;Arruda et al, 2010;Diniz et al, 2007;Hayden et al, 2008;Missiaggia and Grattapaglia, 2006;Ribeiro et al, 2013).…”