(Livak 1999), molecular beacon (Thelwell et al. 2000), and scorpion assay (Whitcombe et al. 1999) are detection systems which are performed in a microtiter plate and based on fluorescent detection systems. Taqman and molecular beacon, both rely on allele‐specific hybridization of oligonucleotide probes to DNA during PCR for allelic discrimination, while scorpion assay can use either allele‐specific PCR or allele‐specific hybridization chemistry for allelic discrimination. All of these microtiter reactions are end point assays and all reagents and genomic DNA are mixed at the beginning, and the fluorescent signal is detected. They are straightforward to accomplish since they do not require a separate preamplification step or intermediary processing.
TaqMan SNP Genotyping
TaqMan SNP genotyping assay utilizes PCR amplification principle for discriminating the samples based on the presence of the SNPs where forward and reverse primers along with Taq polymerase and two SNP specific fluorescent‐labeled probes are used for the amplification. The first probe, specific to allele 1 is labeled with VIC green fluorescent dye, while the second probe is specific to allele 2 labeled with FAM blue fluorescent dye. During the amplification process, probe specific to allele hybridize with template DNA and 5′ to 3′ exonuclease activity of Taq polymerase cleave the probe and remove the fluorescent dye. The dye was further detected by the detector which gives allele‐specific signals. If the sample is homozygous for allele 1, green fluorescence signals from VIC dye are detected by the detector, while the sample is homozygous for allele 2, blue fluorescence signals from FAM dye are detected by the detector. Under the heterozygous condition, there should be a roughly equal signal from both the dyes that produce cyan color signals.
Kompetitive Allele‐Specific PCR (KASP)
KASP assay is a fluorescence‐based genotyping variant of PCR. It utilized allele‐specific primer for amplification and generation fluorescence signals utilizing dsDNA template, two allele‐specific forward primers, common reverse primer where the forward primers are fluorescently labeled having sequences identical to that of 5′ tail of allele‐specific primers. The first round of PCR amplification is carried out using allele‐specific forward primer and common reverse primer. It allows the extension of only perfectly matched primes to result in an amplified product, while mismatched primers remain unamplified. In the third PCR cycle, fluorescence‐labeled forward primer and common reverse primer bind to PCR‐amplified products and extend the amplification reaction. During the primer annealing process quencher molecules present in complementary strands goes away and fluorescence starts emitting which are further detected by the detector.
rhAmp SNP Genotyping
rhAmp SNP genotyping is a PCR assay that uses a unique two‐enzyme system coupled with RNA–DNA hybrid primers to detect the target SNPs. It required a double‐stranded DNA template, two allele‐specific forward primers containing RNA base and blocking moiety at 3′ end, common reverse primer containing RNA base and blocking moiety at 3′ end, RNase H2 enzyme, universal forward primer and universal probe having fluorescence‐labeled at 5′ end and quencher molecule at 3′ end. It involves an allele‐specific amplification process, where allele 1 specific forward primer 1 perfectly binds with SNP site and common reverse primer binds at the 5′ end of the complementary strand. RNase H2 binds cleaves at the RNA base of primer and removes blocking moiety, which allows extension of primers and amplification of both the strand. In the subsequent amplification cycle, the universal primer and universal probe 1 site merged into an amplified product. In PCR cycle 3, universal probe 1 and universal primer 1 bind to the PCR cycle 2 amplified product. Universal forward primer start extension, 5′ to 3′ exonuclease activity of Taq polymerase cleaves the probe and removes fluorescence which was detected by the detector. When allele 2 specific primer 2 binds to the template DNA which generates a universal prob 2‐binding sites labeled with different fluorescence molecules. In this way, different genotypes were clustered based on fluorescence signals detected.
2.2.2.1.3 Hybridization‐based Platforms or Array‐based Methods
Hybridization methods exploit differences in dsDNA thermal stability to discriminate between perfectly matched and mismatched target‐probe combinations in order to achieve allelic discrimination. In the array‐based system, customized probes from the flanking sequence of the SNP are used for genotyping. For a particular locus, the number of probes to be used depends on the number of alleles present. SNPs are usually biallelic in nature, hence mostly two probes distinguishing both alleles are used for each locus. The probes in the SNP array are hybridized with DNA samples to determine the specific alleles of all SNPs on the array for the hybridized DNA sample (LaFramboise 2009). This kind of array has been successfully used in several crops like rice (Thomson et al. 2017; McCouch et al. 2017) maize (Unterseer et al. 2014), wheat (Wang et al. 2014), and barley (Bayer et al. 2017). The number of SNP genotyped at one go has been scaled up to 700 k in rice (Mccouch et al. 2016) and 820 k in case of wheat (Winfield et al. 2016).
There are several well‐established genotyping platforms which can be used for customizing a new array, two of which, i.e., Illumina Infinium Beadchip and Affymetrix GeneChip are most widely used. Further, a new concept of customized array which has a highly specific set of probes used to validate known SNPs in subsets of population is emerging nowadays. Both of the above mention genotyping technology are briefly described below
1 Illumina’s Infinium Beadchips: Infinium array was capable of genotyping only 10k–100k SNPs initially which later has been increased up to 1000k SNPs. It includes first a whole‐genome amplification step, followed by hybridization to bead arrays of 50 bp long capture probes. The locus‐specific sequences include an allele‐specific 3′ terminal base. An allele‐specific primer extension reaction is used to incorporate biotin‐labeled nucleotide‐based or single‐base primer extension step detection systems for positive detection.
2 Affymetrix GeneChip: The GeneChip assays are based on allelic discrimination by direct hybridization of genomic DNA to allele‐specific oligonucleotides (25 mers probes). These oligonucleotides represent all allelic combinations for the particular loci, i.e. perfect match or mismatch probes to each SNP. The range of SNPs to be genotyped ranges from 10k to 2000k SNPs. As gene chip requires high‐quality DNA, so for GeneChip assays to work efficiently, the complexity of the genomic DNA must be reduced through digestion with restriction endonucleases and fractionation. In previous years, researchers used a high‐density oligonucleotide probes array to genotype the entire genome, paving the way for genome‐wide association studies (GWAS) (Kennedy et al. 2003; Matsuzaki et al. 2004; McGall and Christians 2002). Affymetrix GeneChip array contains a large number of synthetic fragments (25‐mer) probes immobilized on a solid substrate. First, the denatured ssDNA are hybridized with these probes, which takes place in a highly specific manner as noncomplementary alleles will not hybridize. Subsequently, the fragments of noncomplementary strands are washed away. The hybrid strands containing probes are stained and then the gene chip is subjected to the CCD imaging device for scanning. Each SNP is represented by a probe set that contains multiple probe pairs. The probe pairs differ in the location of SNP within the oligonucleotide sequence (5 location’s probes are selected). For each position, probes are included from the sense and antisense strand. Hence, the total probes for each allele are 40 probes per SNP.
Although DNA array‐based genotyping has completely revolutionized the field of RNA sequencing and expression analysis, but comparably it has lesser use for SNP genotyping mainly because of the difficulty in obtaining a good signal/noise ratio in allele‐specific hybridization. The resolution of discrimination between completely matched/mismatched oligonucleotides in the array is totally based on high‐throughput fluorescence detection system which is further complicated with an increase in the number of SNPs to be genotyped under a single condition. This specificity compromises genotype call rates and accuracy.
