Secondary‐Structure‐Inducible Ligand Fluorescence Coupled with PCR

Abstract

Single-stranded DNA can change its structure dynamically in response to the presence of a complementary strand. The large structural change from the hairpin secondary structure to the double-stranded form leads to the chemistry of molecular beacons (MBs), or hairpin DNA oligomers having both a fluorescent and a quenching chromophore in the hairpin stem.[1] MBs can be used to report the presence of complementary strands by measuring the increasing fluorescence because of the decreasing energy transfer efficiency. Most DNA probes for fluorescent detection are covalently bound to fluorophores to make energy transfer effective. [1, 2] We have focused on an alternative way to link the fluorescence change with the structural changes of DNA that are induced during polymerase chain reaction (PCR) amplification.[3] We describe herein the chemical concept of noncovalent fluorescent DNA labeling by ligand binding to the secondary structure, which allows us to monitor PCR progress by measuring the change in fluorescence emitted from ligand-primer complexes in a homogeneous solution. This concept is not only complementary to that using fluorescent dye bound selectively to PCR product duplexes, [4] but it also expands the possibility of real-time PCR.The concept of DNA labeling by secondary-structureinducible ligand fluorescence is shown in Figure 1. PCR primers are labeled with a hairpin tag containing cytosine bulges (C-bulges). The molecule 2,7-diamino-1,8-naphthyridine (DANP) binds to a C-bulge in its protonated form (DANPH + ) and emits fluorescence at 430 nm with a 30 nm bathochromic shift from the fluorescence of free, unbound DANP.[5] We hypothesized that, as the PCR progresses, the hairpin tag will dissolve and be transformed into a duplex, thus resulting in the loss of the DANP binding site and a decrease in the fluorescence at 430 nm. Toward this end, we investigated the hairpin tags. These tags should identify the DANP binding site without disturbing the fluorescence efficiency, should not interfere with the PCR, should be transformed effectively into the duplex during PCR, and should be applicable to diverse primers. First, the sequence flanking the C-bulge producing the highest fluorescence intensity was investigated by measuring DANP fluorescence with duplexes having a C-bulge flanked by A-T and T-A base pairs. The G-C and C-G base pairs were omitted from the flanking base pairs because of their quenching of DANP fluorescence.[5b] The characteristic spectrum with a broad single peak at 430 nm was obtained for the A_A/TCT sequence. The relative fluorescence intensity at 450 nm for the four C-bulge duplexes and a fully matched duplex are shown in Figure S1 in the Supporting Information. The C-bulge in the A_A/TCT sequence enhanced DANP fluorescence by 7.6-fold compared with the fully matched duplex and by 82-fold compared with free, unbound DANP.We then designed hairpin tags comprising one to three A_A/TCT units separated by three to five base pairs and a T4 hairpin loop (Table 1). The intensity of DANP fluores...

Synthesis of 1,8-Naphthyridines by the Ionic Liquid-Catalyzed Friedlander Reaction and Application in Corrosion Inhibition

“…1,8-Naphthyridine and its derivatives exhibit excellent optical performance, − various biological activities, and flexible coordination ability, − which led them to be widely used in many fields. As a kind of fluorescent biological probe, they have the characteristic of recognizing specific DNA sequences; − good catalytic activity and thermal stability can be observed when they are used as a ligand for metal ions. − Meanwhile, a large number of 1,8-naphthyridyl compounds own significant anti-inflammatory and antibacterial activity; especially, as anticancer agents, they have shown promising potential. − Because 1,8-naphthyridyl derivatives have a broad range of applicability in many fields, there has been increasing attention toward developing efficient methods for 1,8-naphthyridine synthesis.…”

Competitive Allele‐Specific Hairpin Primer PCR for Extremely High Allele Discrimination in Typing of Single Nucleotide Polymorphisms

“…Because dsDNA can be produced from both matched and mismatched primer-template complexes, diagnosis with AS-PCR by detecting dsDNA has some intrinsic ambiguity. To address this second issue, we have used primer labeling [30][31][32] and have reported on a hairpin primer PCR (HP-PCR) method, [33] which monitors the progress of PCR by detecting not the producing dsDNA but the amount of primer consumed (or remaining). In the HP-PCR method, the primers are labeled with a hairpin tag containing a cytosine bulge (Cbulge).…”

“…CPs are one or two nucleotides longer than HP except for the hairpin tag, and this tag is universal, as reported in our previous paper. [33] Therefore, primer design is quite simple and practical. The power of CPs in allele-specific HP-PCR was confirmed by SNP typing of the cytochrome P450 gene 2C9*3.…”