Читать книгу Genotyping by Sequencing for Crop Improvement - Группа авторов - Страница 45

PCR‐based primer discrimination method simply exploits the accuracy of polymerase to exclude nonspecific nucleotides during extension. In this method, two forward primers are designed from the locus differing in the genotypes while a common reverse primer is used. Both forward primers are specific for their respective alleles. Two different reactions are set for a sample where each reaction contains a different forward primer. Amplification will take place only in any one of these reactions otherwise both DNA is homozygous for the concerned loci.

Taqman (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.