The base-quenched probe method for detecting single nucleotide polymorphisms (SNPs) relies on real-time PCR and melting-curve analysis, which might require only one pair of primers and one probe. At present, it has been successfully applied to detect SNPs of multiple genes. However, the mechanism of the base-quenched probe method remains unclear. Therefore, we investigated the possible mechanism of fluorescence quenching by DNA bases in aqueous solution using spectroscopic techniques. It showed that the possible mechanism might be photo-induced electron transfer. We next analyzed electron transfer or transmission between DNA bases and fluorophores. The data suggested that in single-stranded DNA, the electrons of the fluorophore are transferred to the orbital of pyrimidine bases (thymine (T) and cytosine (C)), or that the electron orbitals of the fluorophore are occupied by electrons from purine bases (guanine (G) and adenine (A)), which lead to fluorescence quenching. In addition, the electrons of a fluorophore excited by light can be transmitted along double-stranded DNA, which gives rise to stronger fluorescence quenching. Furthermore, we demonstrated that the quenching efficiency of bases is in the order of G > C ≥ A ≥ T and the capability of electron transmission of base-pairs in double-stranded DNA is in the order of CG[combining low line] ≥ GC[combining low line] > TA[combining low line] ≥ AT[combining low line] (letters representing bases on the complementary strand of the probe are bold and underlined), and the most common commercial fluorophores including FAM, HEX, TET, JOE, and TAMRA could be influenced by bases and are in line with this mechanism and regularity.

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<b><i>Introduction:</i></b> The molecular biology detection technology of the human <i>ABO</i> blood group system makes up for the limitations in many aspects compared with conventional serological typing technology. This study aimed to establish a new method to identify seven common <i>ABO</i> alleles (<i>ABO</i>*<i>A1.01</i>, <i>ABO</i>*<i>A1.02</i>, <i>ABO</i>*<i>A2.01</i>, <i>ABO</i>*<i>B.01</i>, <i>ABO</i>*<i>O.01.01</i>, <i>ABO</i>*<i>O.01.02</i>, and <i>ABO</i>*<i>O.02.01</i>) by two-dimensional polymerase chain reaction (2D PCR). 2D PCR can identify multiple target genes in a closed test tube by labeling specific primers with tags homologous to the sequence of fluorescently labeled probes, and melting curve analysis is performed after the fluorescent probes are hybridized with tag complementary sequences in PCR-specific products. In this study, 2D PCR and PCR sequence-specific primer (PCR-SSP) were combined to discriminate different alleles in a single reaction, which has the characteristics of high throughput, and compared with other typing techniques; the typing results can be obtained without additional operations. <b><i>Methods:</i></b> The <i>ABO</i>*<i>A1.01</i> allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common <i>ABO</i> alleles were designed on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. Single nucleotide polymorphism sites for identifying seven alleles were detected in FAM and HEX channels, respectively. Two hundred sixty DNA samples were enrolled for rapid <i>ABO</i> genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The <i>Kappa</i> test was used to analyze the agreement of the methodologies. <b><i>Results:</i></b> These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including <i>ABO</i>*<i>A1.01/A1.02</i>, <i>ABO</i>*<i>A1.01/O.01.01</i>, <i>ABO</i>*<i>A1.01/O.01.02</i>, <i>ABO</i>*<i>A1.02/A1.02</i>, <i>ABO</i>*<i>A1.02/O.01.01</i>, <i>ABO</i>*<i>A1.02/O.01.02</i>, <i>ABO</i>*<i>B.01/B.01</i>, <i>ABO</i>*<i>B.01/O.01.01</i>, <i>ABO</i>*<i>B.01/O.01.02</i>, <i>ABO</i>*<i>O.01.01/O.01.01</i>, <i>ABO</i>*<i>O.01.01/O.01.02</i>, <i>ABO</i>*<i>O.01.02/O.01.02</i>, <i>ABO</i>*<i>A1.01/B.01</i>, <i>ABO</i>*<i>A1.02/B.01</i>, and <i>ABO</i>*<i>B.01/O.01. v</i> (containing a rare <i>ABO*O</i> allele, based on the sequencing results). The <i>Kappa</i> test showed completely consistent results for 2D PCR and Sanger sequencing (<i>Kappa</i> = 1). <b><i>Conclusion:</i></b> The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.