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...

Allele-specific polymerase chain reaction (AS-PCR) [1][2][3][4][5][6][7][8][9][10] is one of the most convenient and direct methods for single nucleotide polymorphism (SNP) typing, [11][12][13] and is based on the selective elongation of the primer that matches the template sequence. However, the discrimination of two alleles by allele-specific primers is not always complete because of subtle differences in the thermodynamic stability between matched and mismatched primer-template complexes. Elongation of the primer from the mismatched primer-template complex will eventually match the template to the formerly mismatched primer. Because of the exponential amplification of the analyte DNA by PCR, formation of a matched template for the mismatched primer significantly reduces allele specificity. Although methods to improve allele specificity have been developed by using primer design, [14][15][16][17][18][19][20][21] allele specific blocking [22][23][24] and clamping, [25,26] and engineered polymerase, [27][28][29] a complete solution remains to be devised.Another issue in AS-PCR is the fact that diagnosis with AS-PCR usually relies on the detection of amplified double-stranded DNA (dsDNA). 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). The fluorescent molecule 2,7-diamino-1,8-naphthyridine (DANP; Scheme 1 A) bound to the C-bulge and emitted fluorescence at around 430-450 nm with a 30 nm bathochromic shift from the fluorescence of free, unbound DANP. The hairpin structure was unfolded and converted into the double-stranded form by DNA polymerase with concomitant release of the bound DANP from the hairpin tag, resulting in a decrease in the fluorescence at 450 nm. The fluorescence intensity was monitored at 450 nm to quantify consumption of the HP in this HP-PCR method. [34,35] Exploiting this ability of HP-PCR to detect the amount of primer, as compared with conventional PCR, which detects the product dsDNA, we here describe our approach to solve the [a] Dr.

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...

No abstract

Single-cell analysis is important to understand how individual cells work and respond at the cell population level. Experimental single-cell isolation techniques, including dilution, fluorescence-activated cell sorting, microfluidics, and micromanipulation, have been developed in recent decades. However, such applications typically require large cell populations and skilled professionals. Additionally, these methods are unsuitable for sequential analysis before and after cell isolation. In this study, we propose a method for target cell isolation using automated infrared laser-mediated disruption of pollen grains in pollen populations. Germination of the target pollen was observed at the same location as that before laser irradiation, and germinated pollen grains were enriched in the cell population. Pollination of laser-irradiated bulk pollen populations also showed that the target pollen preferentially germinated on the stigma. This method is expected to facilitate physiological analyses of target cells at the single-cell level and effectively produce seeds derived from target pollen.