TSP was developed to support the deployment of single-marker SNP genotyping methods in the laboratory and to reduce bottlenecks in genotyping throughput that can be caused by the requirement to optimize individual assays. TSP employs a biphasic PCR mechanism that simplifies the design of single-marker SNP genotyping assays, reduces requirements for individual assay optimisation, and increases assay specificity and genotyping accuracy. It should be possible to directly incorporate TSP into the design of assays for most current single-marker SNP genotyping methods. TSP assays are robust and support the amplification of SNP genotyping products for both the presence and absence of an allele within the range of 60 to 500 bp. Flexibility for genotyping product size provides maximal compatibility for the separation and detection of SNPs on a variety of size separation matrixes such as agarose gel, and a range of dedicated instruments such as those used for capillary electrophoresis and melt curve analysis. TSP provides further opportunities to increase genotyping throughput by assay automation, and by supporting automated data acquisition and genotype calling.
The biphasic PCR mechanism of TSP creates a method for multiplex PCR that is performed under standardized conditions, and therefore does not require optimisation of individual assays. In SNP genotyping, TSP enables the sequential amplification of a target sequence harboring a polymorphism, followed by interrogation of the SNP in a single-step assay. The biphasic mechanism allows nested PCR to occur as two distinctly separate reactions within the same vessel, in which all of the primers required for amplification are present. This is achieved through a difference in melting temperature between the two sets of primers used for the nested PCR such that the participation of each primer set can be specifically controlled by annealing temperature. The separation of the two phases of amplification was shown using QPCR (Figure ) by the differences in cycle thresholds among primer sets and the reduction in fluorescent intensity when both sets of primers are present in the reaction, reflecting the amplification of the shorter genotyping product.
In TSP assays, separating the participation of the different primer sets by their annealing temperature provides several advantages for SNP genotyping. With fixed primer design parameters, SNP genotyping assays can be developed rapidly, and it is possible to increase the flexibility and throughout of genotyping by assaying different SNPs in the same reaction plate. The fixed primer design parameters also minimize the need to optimise individual SNP genotyping assays, since TSP has been optimized to ensure that robust biphasic PCR occurs every time. By partitioning the step for amplification of the target sequence from the step for interrogation of the SNP, primers for each stage of the assay can be optimally designed. This can improve assay specificity because primer design parameters for the first stage of the SNP genotyping process are not compromised in specificity for optimal parameters that may be different for the next stage of amplification. In other single-marker SNP genotyping methods, where multiple primers sets designed with similar melting temperature are present in the reaction, undesirable primer-primer interactions and non-specific amplification from the genomic template can result unless careful consideration is given to primer design and assay optimisation.
The application of TSP for single-marker SNP genotyping using allele-specific PCR and HRM analysis was used in the present study to illustrate the advantages of TSP over published methods based on these assay chemistries. Allele-specific PCR and HRM assays are deployed in many laboratories for endpoint SNP genotyping due to their relatively low assay cost and flexibility for meeting the changing demands of genotyping throughput that is associated with many genetic research and diagnostic applications.
Several allele-specific PCR methods for SNP genotyping are reported including ARMS, PASA, and modifications of these methods such as tetra-ARMS and bi-directional PASA (bi-PASA) [3
]. These methods are based on the annealing and extension of a primer that is specific to the allele of interest. They commonly employ two flanking primers to amplify the target sequence harboring the polymorphism and a third primer adjacent to the polymorphism to interrogate the SNP. The addition of a fourth primer to the assay allows the interrogation of both SNP alleles and therefore the presence of heterozygosity to be detected in a single assay. The main limitation to these methods, especially for assays based on the four-primer system, is a complicated primer design process (due to the requirement to design primers that do not result in undesirable primer-primer, or primer-template interactions that comprise assay specificity), the need to optimise individual assays to achieve allele-specific amplification (usually by individual adjustment of the concentration of each primer in the assay), and variable amplification efficiency due to differences in product length (caused by PCR competition effects). Significant effort has been invested into ways to improve the specificity of allele-specific PCR through modification of the allele-specific primers including the introduction of a secondary mismatch near the 3'-terminus of the primer, and the use of modified nucleotides such as LNA and phosphothioate linkages [20
], but these add to the complexity of assay design and cost.
The configuration of TSP for allele-specific PCR simplifies assay primer design and reduces the requirement for optimisation of individual assays. The biphasic PCR mechanism avoids undesirable primer-primer interactions between the different sets of primers by separating their participation into different stages of the reaction. It also prevents undesirable participation of the NLS primers in the early stages of the reaction, which can compromise assay specificity and genotyping accuracy. This latter feature reduces the possibility of mis-priming, which can occur when amplifying and enriching a SNP directly from genomic template. Mis-priming compromises genotyping accuracy because PCR product for the alternate SNP allele can accumulate even in the absence of that allele, a phenomenon known as allele leakage. The ease for designing allele-specific PCR assays using TSP was demonstrated by developing 87 TSP markers, whose assays are performed under identical reaction conditions. Only seven of these markers showed SNP allele leakage that compromised genotyping accuracy, and in each case this was resolved by targeting the alternate SNP allele. High genotyping accuracy was demonstrated by 100% concordance of the 11,232 TSP genotypes generated in a blinded study with an independent genotyping method. Moreover, genotyping accuracy was maintained across samples with a range of DNA quality, indicating the tolerance of TSP to samples prepared using different DNA extraction methods. The only factor found to affect genotyping accuracy was overloading the assay with DNA (> 100 ng), a result consistent with what has been found for other allele-specific PCR genotyping methods, for which a low DNA starting concentration is best [14
TSP confers advantages over current allele-specific PCR methods for endpoint SNP genotyping. In addition to the benefits of speed of assay design and genotyping throughput, TSP provides the flexibility to achieve codominant genotyping whilst targeting only one allele for primer design, since the amplification of an allele-specific, alternate allele, or both genotyping products indicates the presence of homozygous and heterozygous alleles (Figure ). It also provides a mechanism to distinguish between the absence of the target allele and a failed reaction, since genotyping products are always produced. Perhaps the most important advantage of TSP for allele-specific PCR is flexibility for assay design. In a separate study, we showed the same assay specificity and genotyping accuracy could be achieved using a three-primer system, in which only one nested locus-specific primer is present [22
]. In these assays, the nested locus-specific reverse primer was removed. The advantage of a three- versus four-primer TSP design for allele-specific PCR depends on the sequence context flanking the SNP and the requirement for a specific PCR product size for the alternate allele PCR genotyping product, which depends on the platform used for detecting the SNP genotyping products. These TSP assay configurations for allele-specific PCR support rapid, low-cost endpoint SNP genotyping, since they are not reliant on fluorescently-labeled primers or probes and they do not require individual assay optimisation to accurately assign a genotype. Rather, the standardized assay conditions and simple codominant genotyping data output provides opportunities to automate assay setup, data acquisition and genotype calling.
Similarly, the configuration of TSP for endpoint SNP genotyping using HRM illustrates the benefits of incorporating TSP into the design of SNP genotyping methods that are based on detecting changes to the physical properties of DNA. Detection of the polymorphism by HRM relies on the differential melting of amplicons based on a single base difference. The detection sensitivity is affected by both the size of the PCR fragment and the position of the SNP within the amplicon [23
]. However, it can be difficult to capture the polymorphism at an optimal position within the PCR fragment to achieve maximal detection sensitivity. This can result from unfavorable flanking sequence composition and high sequence similarity between related genes; a problem often encountered for the assay of SNPs in multi-gene families and duplicated genes. In such instances, TSP can provide an advantage for endpoint SNP genotyping, since the ability of the biphasic mechanism to perform nested PCR in a single-step assay eliminates the requirement for a separate assay to preamplify the target sequence harboring the SNP. By enriching the target sequence from the genomic template in the first phase of the TSP reaction, a set of nested locus-specific primers designed for maximal detection sensitivity can be used without concern for interference by other factors that would otherwise confound SNP detection. The design of these TSP assays, however relies on the ability to design LS primers for specifc amplification of the gene of interest. In the present study, all of the TSP genotyping assays designed for endpoint HRM detection of the SNP produced robust amplification of the target sequence as a single genotyping product (Figure ), demonstrating the fidelity required for accurate SNP genotyping. TSP could also be useful in SNP genotyping methods that are based on enzymatic modification, as these also require that the SNP be positioned appropriately within the PCR fragment (in order to produce scorable restriction fragments).
TSP was developed and optimised using genomic DNA from cultivated barley (Hordeum vulgare
L.), an agriculturally important crop with a 5,300 Mb diploid genome about twice the size of the human genome. Assays were performed using between 20 and 100 ng of genomic DNA without loss of sensitivity or specificity. Adapting TSP for use in another species, or with a different amount of starting genomic template only requires optmisation of the PCR cycle number in the first phase of the assay. Once the optimal cycle number is determined, all other TSP primer design parameters and reaction conditions remain the same. TSP has been successfully deployed for SNP genotyping in zebrafish [24
], human [25
] and mouse (unpublished data) using allele-specific PCR. These assays were performed using the TSP cycling conditions described for barley, and between 20 and 50 ng of genomic DNA as starting template.
Most SNP genotyping methods have been developed to assay diploid organisms. In polyploid organisms, including many plants, genotyping is complicated by the presence of two or more gene copies in the nucleus. Selective PCR amplification using primers specific to one or another copy of the duplicated locus [26
] is frequently used as a strategy to overcome this complication. However, this approach cannot be easily scaled-up and used for developing high-throughput genotyping assays. A potential advantage of TSP is its ability to address the challenges of SNP genotyping in polyploid genomes by enabling both the amplification and interrogation of a specific gene copy harboring a SNP. The successful application of a single-step assay in a polyploid system requires complete separation of the participation of the primer set for genome-specific (or gene copy-specific) amplification from the participation of the primers for interrogating the SNP. The gene copy harboring the SNP must be enriched substantially over other copies of the gene before the allele-specific (nested) primers are able to participate. It is only then that amplification from these allele-specific primers can be diagnostic for the presence-absence of the target SNP. The utility of TSP for SNP genotyping in polyploid plant species using allele-specific PCR and HRM detection methods is now being investigated in our laboratory.