A new technology has been developed for homogeneous, sensitive, rapid, and cost-efficient analysis of SNPs and mutations in genomic DNA. The GALIOS™ Genotyping System (Evotec Technologies GmbH, Düsseldorf, Germany) combines the high specificities of seminested PCR and allele-specific amplification, concurrently performs amplification and allele-specific labeling, and simultaneously interrogates both alleles of a given bi-allelic SNP in a ''one-tube'' reaction. Moreover, the GALIOS™ Genotyping System is fully generic and does not require the expensive and time-consuming synthesis of specialty primers or probes. Figure 1 illustrates the GALIOS™ principle of bi-allelic SNP scoring in a one-tube reaction. The reaction mix contains four target-specific and two universal primers; one pair of gene-specific amplification primers, two types of seminested, allele-specific diagnostic primers containing different universal sequence tails at their 5'-ends, and two types of fluorescent universal detection primers tagged with fluorescence dyes excitable at 543 nm (TAMRA) or 633 nm (EVOblue™), respectively. The tailed diagnostic primers have identical sequences, except for the 5'-tail portion and at least one internal nucleotide, which is complementary to the corresponding nucleotide of the respective allele. During a GALIOS™ reaction, the high-concentrated amplification primers dominate the early PCR cycles and amplify the genomic region of interest independent of the allele. In contrast, the low-concentrated diagnostic primers are extended predominantly in later PCR stages and lead to allele-specific generation of universally tailed PCR products. Upon progress of the
Principle of GALIOS
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