Introduction
High-throughput technologies for nucleotide sequence analysis and detection of sequence variation have been increasingly used for plant and animal genotyping, forensics, genetic medicine, and other fields of genetic testing. The most frequently encountered sequence variations in any genome are single-nucleotide polymorphisms [SNP] [Clevenger et al., 2015]. The primary consideration for selecting a marker type for genotyping is the information content of the polymorphisms and the informative capacity of the test. In this regard, SNPs are very effective markers. A variety of SNP genotyping methods are available, and new methods appear regularly with the aim of reducing cost and increasing throughput. PCR can be adapted for rapid detection of single-base changes in genomic DNA by using a family of closely related methods, such as allele-specific PCR [AS-PCR] [Ugozzoli and Wallace, 1991; Bottema and Sommer, 1993; Spierings et al., 2006], PCR-amplification of specific alleles [PASA] [Sommer et al., 1989; Sarkar et al., 1990], allele-specific amplification [ASA], and amplification refractory mutation system [ARMS] [Newton et al., 1989; Nichols et al., 1989; Wu et al., 1989]. All these methods use specifically designed PCR-primer sets containing allele-specific primers [ASP] in which the allele specificity is determined by the base at or near the 3′ end. In principle, AS-PCR assays can be developed to analyse almost any allelic variation [Sommer et al., 1989; Ye et al., 1992].
There currently exists an enormous number and variety of established methods applicable for SNP analysis and genotyping based on distinctly different platforms and approaches [Kim and Misra, 2007]. FRET [Fluorescence Resonance Energy Transfer] is one such method and is based on dual fluorescence that is quantified during ligation. Another method is allele-specific PCR with SNP targeting [Didenko, 2001; Kaur et al., 2020]. Single-plex PCRs for SNP analysis, such as TaqMan [Life Technologies, USA] and SimpleProbe [Roche Applied Science, USA] have been described as valuable additions for marker-assisted selection in plant breeding [Makhoul et al., 2020; Della Coletta et al., 2021]. Further improvements for AS-PCR include using fluorescence to detect the amplification of a specific allele. Kompetitive Allele Specific PCR [KASP] [LGC Biosearch Technologies, Teddington, UK] and PCR Allele Competitive Extension [PACE] [Integrated DNA Technologies, Inc.] genotyping represent developments to the original AS-PCR approach. These techniques use fluorescence resonance energy transfer [FRET] for signal generation and both allow accurate bi-allelic discrimination of known SNPs and insertion-deletion polymorphisms [InDels]. The KASP method has great potential for expanded utilization, as it requires only a slight modification to commonly used procedures for designing ASPs, offers convenient FRET detection, and master-mixes are commercially available [Wang et al., 2015; Ryu et al., 2018; Brusa et al., 2021]. In KASP, PCR amplification is performed using a pair of allele-specific [forward] primers and a single common [reverse] primer. In addition to the common-core primers, the reaction mixture is supplemented with a FRET cassette. This is a duplex of two synthetic complementary oligonucleotides; one is labelled with a fluorescent dye and the other carries a fluorescence quencher. Further, each ASP has a unique 5′ terminal extension [tail], which is complementary to the sequence in the FRET cassette. The oligonucleotides in the FRET cassette are modified such that they do not participate in polymerase-mediated extension steps. The dye-labelled oligonucleotide is capable of annealing to the reverse-complement of the tail sequence in PCR fragments containing one selected allele. During amplification of the allele with participation of the tailed ASP, an amount of DNA increases to which the dye-labelled component anneals. This disrupts the integrity of the FRET cassette and the fluorescent dye is spatially separated from the quencher and thus able to emit fluorescence. Unlike other PCR-based genotyping assays, KASP/PACE requires no labelling of the target-specific primers/probes, which provides additional flexibility in the assay design. Both methods use two reporting cassettes. If a genotype at a given SNP is homozygous, only one of the two possible fluorescent signals will be generated. If the genotype is heterozygous, both fluorescent signals will be generated. KASP/PACE technology is especially suitable for high-volume screening projects, such as in plant breeding. KASP/PACE technology has a key feature, which is utilizing a universal FRET cassette reporter system that eliminates the need for dual-labelled probes. Commercial companies produce PCR additives, called master mix, which contains one or more ‘universal’ FRET cassette[s]. In theory, this additive can be used to upgrade existing AS-PCR assays to KASP/PACE, provided that new ASPs are used that have the described cassette-specific tails. For example, a protocol upon which this work is based, uses chemistry consisting of the following two parts. These are the assay mix [template-specific, contains target DNA, two ASPs [for binary SNP] and a single reverse primer] and master mix [not template-specific, combines all reagents required for PCR and in addition contains two different FRET cassettes, one cassette labelled with FAM dye and the other with HEX dye]. Each ASP carries a unique tail sequence that corresponds to a FRET cassette. In other FRET AS-PCR methods, such as Amplifluor [Rickert et al., 2004; Fuhrman et al., 2008] and STARP [Rasheed et al., 2016; Long et al., 2017; Li et al., 2019; Wu et al., 2020], these two parts are completely separated into non-labelled ASPs such that the last base of the primer’s 3′ end base is positioned on the SNP site, and labelled universal probes [UPs] that carries a fluorophore at the 5′ terminus and a quencher attached in the middle of the universal probes [Nazarenko et al., 1997]. Simple and inexpensive ASPs can be designed and ordered for each SNP separately, while the relatively expensive UPs with fluorophores and quenchers are ordered just once for a stock that can be used over a very long time in many different SNP analyses. The principle of ASP-UP is similar to that of Molecular Beacons, with the addition of specialized identical “tags” at the 5′ end of the ASP and the 3′ end of the UP. In this particular case, ASPs are slightly longer [Myakishev et al., 2001]. In KASP-related methods, a set of non-labelled ASPs includes two forward primers and a single common reverse primer that act on a competitive basis in conjunction with one of two corresponding UPs with “hair-pin” FRET structures that end with either FAM or HEX/VIC fluorophores. This approach allows for great flexibility in assay design, which translates into a higher overall success rate for SNP genotyping and detection of InDels. This principle of separated ASPs and UPs is used in various methods, including commercially produced Amplifluor [Merck KGaA] and KASP markers [LGC Biosearch Technologies] for fluorescent signal generation that enable bi-allelic discrimination and genotyping of SNPs or InDels.
Developing high-throughput, multiplex genotyping assays require using computerized approaches to design primers and probes and select reaction conditions. In recent years, several software tools have been developed to aid AS-PCR assay development, including GSP [Wang et al., 2016], PrimerSearch-EMBOSS [//emboss.open-bio.org/rel/rel6/apps/primersearch.html], WASP [Wangkumhang et al., 2007], PolyMarker [Ramirez-Gonzalez et al., 2015], KASPspoon [Alsamman et al., 2019], and PUNS [Boutros and Okey, 2004]. However, not all of these programs are readily available or broadly applicable; some are no longer actively updated whilst others are extremely narrow in their applicability.
Here we describe a software tool and a modified KASP method that further increases the convenience and power of the genotyping protocol. We have named this allele-specific quantitative PCR [ASQ]. The software facilitates the development of assays using KASP and PACE technology and works with SNP and InDel polymorphisms. For all polymorphisms taken into the study, the program produces the thermodynamically optimal combination of ASPs for single-plex or multiplex assays. During ASP design, a user can add a tail sequence [which can be a custom sequence] or a sequence that matches a commercially available aster mix [e.g., manufactured by LGC Biosearch Technologies, or Merck KGaA]. If a commercial FRET cassette is not optimal, the program allows use of custom FRET cassettes. The program also computes the optimal reaction conditions to perform KASP. This software will be most useful for multiplex genotyping in a high-performance environment. Furthermore, potential uses are not limited to genotyping. The program can produce tests for selecting mutants, e.g., after genome editing or gene knockout [Lee et al., 2016]. Other possible applications include analysis of genetic variations in microorganisms, strain identification, detecting genetic markers of resistance or virulence, and tracing pathogens in epidemiology and medical diagnostics. The ASQ method described here is a tool for assessing the relative amount of different allelic variants in a sample. This method can be useful for a variety of tasks. For example, ASQ can be used to measure a fraction of admixed GMO material in samples of agricultural products or to measure a portion of malignant cells in samples from cancer patients. Distinctive features of the presented program make it unique in its class. These features include computing of multiplex reactions for simultaneous identification of up to four alleles [e.g., 3-state or 4-state polymorphisms]; use of input polymorphisms of mixed type [overlapping SNP and InDel sites]; and absence of restrictions on the length difference between InDel alleles during genotyping of InDels. The program automatically calculates primers for possible amplification in both directions from the polymorphic site. The program allows the user to include in the protocol commercially available master mixes and FRET cassettes or to create unique FRET cassettes and use custom reagents in designing other assays. The software described herein is integrated into and works inside the FastPCR environment. FastPCR is an integrated software space for developing PCR-based processes. The FastPCR program, including the presented AS-PCR-designing tool, may be used online or downloaded and used in Microsoft Windows [Kalendar et al., 2011; Kalendar et al., 2017] and is freely available at //primerdigital.com/tools/. This AS-PCR tool simplifies and automates design of genotyping assays, resulting in a greater likelihood of success.
Materials and Methods
Selection of Highly Informative SNP Candidates
SNP data in the SNPforID browser [//spsmart.cesga.es/snpforid.php] were employed in the present study for SNP genotyping of humans in forensic studies. The allelic frequencies were used for screening to select highly informative SNP candidates. As markers with even allelic distributions have high observed heterozygosity and are more informative, 7 of the SNPs were selected with a common 40:60-60:40 allelic distribution in European and Central Asian populations. All of the selected markers are located on autosomal chromosomes [Supplementary Table S1]. The marker also known as “C/T[-13910]” located in the MCM6 gene but with influence on the lactase LCT gene [rs4988235] is one of two SNPs that is associated with the primary haplotype associated with hypolactasia, or more commonly known as lactose intolerance in European populations.
Sample Preparation
This work was discussed by the institutional review board and was approved by the ethical committee of the Center for Life Sciences, National Laboratory Astana, Nazarbayev University [protocol #21, 10 October 2017]. Institutional written informed consent about nationality declaring, DNA extraction and for further investigation was signed and obtained from the participated individuals.
Informed consent was obtained for each participant. DNA was extracted using QIAamp DNA Blood Mini Kit [Qiagen, Hilden, Germany]. The extracted DNA pellet was diluted in TE buffer [10 mM Tris pH 8.0, 0.1 mM EDTA], and DNA concentration was measured with a NanoDrop spectrophotometer [Thermo Fisher Scientific, USA]. Quality was assessed using 1 mg of DNA visualised after running in a 1% agarose gel.
Real-Time PCR Analysis
A QuantStudio-7 Real-Time PCR instrument [Thermo Fisher Scientific, USA] and CFX96 Real-Time PCR Detection System [Bio-Rad, USA] were used. These instruments have detection systems with filters for FAM, VIC/HEX, Cy3, Cy5, and ROX fluorophores. While SNP identity calls were made automatically using software accompanying the instruments, amplification curves were checked for each genotype manually for final allele discrimination. SNP genotyping experiments used at least three to eight biological replicates and were repeated three times.
The PCR conditions employed an altered PCR cocktail composition [Table 1]. PCR plates with 96-wells were used with a 15 μl total reaction volume in each well. The PCR mix consisted of the following reagents: 1x OneTaq buffer [total 3 mM MgCl2], 0.2 mM of each dNTP, 0.2 μM of each UP, 0.5 μM quencher oligo, 0.1 μM of each AS primer, 0.3 μM of reverse primer, and 0.5 units of Taq DNA polymerase [NEB]. Half of the PCR volume was genomic DNA, adjusted to 5 ng/μl.
TABLE 1. PCR cocktail mix composition for the proposed ASQ method of SNP genotyping.
The PCR program was optimised and consisted of 95°C, 120 s; 10 cycles of 10 s at 95°C; 20 s at 55°C; 20 s at 68°C; 30 cycles of 10 s at 95°C; 30 s at 68°C; 30 s at 55°C [Table 2]. Fluorescence was monitored during the last step at a second annealing. ASQ was performed to simultaneously detect two alleles in a single tube. Each well was examined for the characteristic fluorescent emissions of both fluorescein [FAM channel] and HEX [HEX channel].
TABLE 2. PCR protocol for genotyping using advanced ASQ method for SNP genotyping.
Genotyping with SNP calling was determined automatically. Each experiment was repeated twice and technical replicates confirmed a confidence of SNP calls.
KASP Assay Design Method
Standard KASP allows genotyping of only two alternative alleles at any specific site. This is because there are two only FRET cassettes, and thus two fluorochromes, present in commercially available kits. However, advances in real-time PCR equipment allow detection of up to 6 spectrally separated dyes.
In addition, there are approaches to overcome physical spectral dye channels and expand the potential of spectral channels to practically detect an unlimited number of independent targets [Marras et al., 2019]. Theoretically, the number of different multiplex targets or alternative alleles that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colours for the identification of each target allele [Marras et al., 2019]. The novel tool described here allow detection of up to 4 spectrally separated dyes [Figure 1]. Moreover, the program calculates even more assay possibilities because the ASPs are searched for on both strands and at both sides from a polymorphism site [Figure 2]. The user has the possibility to use the single most suitable set of primers from the output or, if desired, alternative sets of primers may be used to target the same site. The tool also allows design combinations of tail sequences to create a multiplex PCR targeting several different polymorphic sites simultaneously. There is a need to incorporate flexibility in a primer-designing algorithm to select AS primers, as placing the 3′-terminal nucleotide on the polymorphic site may not be an optimal solution for some sites. Briefly, to achieve optimal amplification, the tool automatically selects the primer length and the position of its 3′ terminus for each ASP. This kind of optimization is also necessary when working with InDels. For example, for a null-site polymorphism [i.e., one or more nucleotide[s] absent in one allele or there are insertions in other alleles], the ASP sequence to target this allele will be positioned such that the primer’s 3′-terminal sequences are sufficient for annealing. Thus, ASP sequences are properly adjusted for each allelic variant. In case of short InDels [