Universal primers for fluorescent labelling of PCR fragments—an efficient and cost‐effective approach to genotyping by fluorescence

Abstract

Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5' universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-specific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.

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

“…However, the use of large sets of SSR markers is limited due to the high cost and the amount of work required for their analysis, as well as the difficulties of automation. Those limitations could be partially overcome by adopting multiplexing strategies (Butler 2005a, b;Missiaggia and Grattapaglia 2006;Blacket et al 2012) that would led to significant savings in time, efforts, and laboratory reagents (Elnifro et al 2000;Raabová et al 2010). Empirical results demonstrated that SSR-based multiplexing and multiloading reduced the cost of PCR reagents up to 50 % and the cost of electrophoresis up to 85 % when compared to genotyping based on single-locus PCR (Masi et al 2003;Merdinoglu et al 2005).…”

“…In spite of these advantages, microsatellite markers present some limitations: mainly the high cost and time-consumed per individual SSR assay (Guichoux et al 2011). In order to increase cost and labor efficiency of SSR analysis, multiplex PCR approaches combined with fluorescence-based analysis and semiautomated allele calling have been developed (Butler 2005b;Missiaggia and Grattapaglia 2006;Blacket et al 2012). These strategies allow to simultaneously amplify more than one locus in a single reaction using multiple primer pairs.…”

Rapid and effective isolation of candidate sequences for development of microsatellite markers in 30 fish species by using kit-based target capture and multiplexed parallel sequencing

“…From the runs, we obtained 126,196, 165,625, and 204,813 Four tail sequences (Blacket et al 2012) were the followings: Tail A, GCC TCC CTC GCG CCA; Tail B, GCC TTG CCA GCC CGC; Tail C, CAG GAC CAG GCT ACC GTG ; Tail D, CGG AGA GCC GAG AGGTG Bold for the F ST value indicates the significant differences using 16,000 permutations (P < 0.05) n number of individuals genotyped, N A number of alleles, R range of observed alleles, H O observed heterozygosity, H E expected heterozygosity *Significant deviation from Hardy-Weinberg equilibrium (P < 0.05) total, respectively. The average sequence length for each sequencing run was 298, 280, and 320 bp, respectively.…”

“…These primer sets were amplified using five multiplex PCR reactions (Table 2). PCR reactions were carried out in 7 μl of final reaction mixture (Sets I and II) or 4 μl (Sets III-V), containing 1 × Type-it Microsatellite PCR kit (Qiagen), approximately 50-250 ng of template DNA, and the forward primer, reverse primer, and universal tail primer in a 1:2:1 ratio (Blacket et al 2012). The final concentration of the forward primers was optimized for each marker (Table 2).…”

“…After all this exclusions, 88 sequences were retained for primer synthesize. To facilitate multiplex PCR, we selected 44 of 88 primer sets based on expected PCR product size and the forward primer of each locus was synthesized with one of four universal tag sequences (Table 2; see also Blacket et al 2012). The four universal tag primers can be combined with four fluorophores to co-amplify multiple loci via multiplex PCR.…”