Single-labeled Probes Research Articles

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations

Results of the steady-state fluorescence, anisotropy and FRET measurements of G-quadruplex formation in the presence of selected cations (Li +, Na +, K +, NEt 4 + and Mg 2+) are reported. Three different fluorescent oligonucleotides with human telomeric sequence labeled with fluorescein (FAM) and tetramethylrhodamine (TAMRA) were investigated: a dual-labeled 21-mer denoted as PSO (Potassium Sensing Oligonucleotide) and two 5′- and 3′- single-labeled probes, FAM-21 and 21-TAMRA, respectively. The fluorescence signal of FAM-21 increased significantly for all systems and the fluorescence enhancement was comparable in magnitude for monovalent cations but it was more pronounced for Mg 2+ cation. This phenomenon was attributed to the protolytic equilibria of FAM affected by the variation in ionic strength. On the other hand, fluorescence of TAMRA was enhanced selectively by Na(I) cation that was explained by the dequenching of TAMRA emission originated from the peculiarity of the basket-type structure of Na(I)-quadruplex. Anisotropy of FAM-21 (but not 21-TAMRA) appeared to be sensitive to the G-quadruplex formation, showing significant increase with an increase in cation concentration and indicating some restrictions in rotational depolarization of FAM. FRET experiments revealed that all tested cations caused quenching of FAM fluorescence in PSO, but only Na + and K + ions produced sensitized emission of TAMRA acceptor. Higher FRET efficiency observed in the presence of sodium ion was attributed to the specific spectral factor and steric interactions in the basket-type Na(I)-quadruplex.

Validating a Rapid Method for Detecting Common Polymorphisms in the APOA5 Gene by Melting Curve Analysis Using LightTyper

The recently identified apolipoprotein A-V gene (APOA5) has been shown to play an important role in hypertriglyceridemia (1). Genetic variation in APOA5 has been consistently associated with plasma triglyceride concentrations in several studies (2)(3)(4). Moreover, some studies have demonstrated additional associations with lipoprotein subclasses, remnant-like particles, and cardiovascular disease risk (4)(5)(6). Several single-nucleotide polymorphisms (SNPs) in the human APOA5 gene have been detected with differing frequencies depending on the population analyzed (7)(8), and Klos et al.(7) have also suggested context-dependent associations in different populations. Overall, 5 common SNPs, −1131T>C, −3A>G, 56C>G, IVS3 + 476G>A, and 1259T>C, have been widely reported. Apart from the 56C>G SNP, the other SNPs are in strong linkage dysequilibrium, producing 3 haplotypes (11111, 22122, and 11211) representing ∼98% of the population in Caucasians (5)(9); therefore, 2 independent APOA5 SNPs (56C>G and −1131T>C) can be analyzed in association studies as indicators of the corresponding haplotypes. The former consists of a nonsynonymous substitution, changing codon 19 from serine to tryptophan (S19W), whereas the latter is a T-to-C substitution 1131 nucleotides upstream of the initiation codon. To date, in most published reports, these SNPs have been genotyped by PCR with restriction fragment length polymorphism (RFLP) analysis (3). A system for high-throughput genotyping using fluorescence melting curve analysis, the LightTyper™ (Roche), has recently become commercially available. This instrument (10) offers higher throughput than the original LightCycler™ (11). The LightTyper is designed explicitly for melting curve analysis to perform rapid, straightforward, reliable allelic discrimination. The system, which uses 384-well plates, provides postamplification genotyping within 10–15 min and performs genotyping automatically. A variety of probe chemistries are compatible for genotyping, including single-labeled probes and fluorescence resonance energy transfer probes (10). SimpleProbes™ are designed to specifically hybridize to a …

The LightTyper TM : High-Throughput Genotyping Using Fluorescent Melting Curve Analysis

Instrumentation, chemistry, and software for high-throughput genotyping using fluorescent melting curves are described. The LightTyper system provides post-amplification genotyping within 10 min using samples in 96- or 384-well microplate formats. The system is homogenous because all reagents are added at the beginning of the reaction and there is no sample manipulation between amplification and genotyping. High-resolution melting curves are achieved by slow and steady heating. As samples are heated, panels of blue light-emitting diodes excite the probes, and fluorescence emission is acquired with a cooled charge-coupled device camera. A variety of probe chemistries are compatible for genotyping on the LightTyper, including dsDNA dyes, single-labeled probes, and fluorescence resonance energy transfer systems. Genotyping is performed automatically, and each sample is given a score reflecting the similarity of the genotype to the standards provided. Standard genotypes can be selected from within the run or imported from other files. Samples and genotypes can be grouped to allow multiple-allele detection on one or many samples. The utility of the LightTyper is illustrated by genotyping samples for the Factor V Leiden mutation and for mutations in the CFTR gene.

Real-Time Multiplex PCR Assays

The ability to multiplex PCR by probe color and melting temperature (Tm) greatly expands the power of real-time analysis. Simple hybridization probes with only a single fluorescent dye can be used for quantification and allele typing. Different probes are labeled with dyes that have unique emission spectra. Spectral data are collected with discrete optics or dispersed onto an array for detection. Spectral overlap between dyes is corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra. Since fluorescence is temperature dependent and depends on the dye, spectral overlap and color compensation constants are also temperature dependent. Single-labeled probes are easier to synthesize and purify than more complex probes with two or more dyes. In addition, the fluorescence of single-labeled probes is reversible and depends only on hybridization of the probe to the target, allowing study of the melting characteristics of the probe. Although melting curves can be obtained during PCR, data are usually acquired at near-equilibrium rates of 0.05–0.2°C/s after PCR is complete. Using rapid-cycle PCR, amplification requires about 20 min followed by a 10-min melting curve, greatly reducing result turnaround time. In addition to dye color, melting temperature can be used for a second dimension of multiplexing. Multiplexing by color and Tm creates a “virtual” two-dimensional multiplexing array without the need for an immobilized matrix of probes. Instead of physical separation along the X and Y axes, amplification products are identified by different fluorescence spectra and melting characteristics.

Chromosomal in situ hybridization with double-labeled DNA: signal amplification at the probe level.

A double-labeling approach was applied to nonisotopic in situ hybridization with individual cosmid and plasmid clones, using digoxigenin or biotin as label and a combination of two separate enzymatic labeling methods. Probe labeling was achieved by nick translation, followed by tailing of the probe by terminal deoxynucleotidyl transferase. The double-labeling method, in conjunction with an improved detection protocol, provides for a higher signal intensity than that obtainable with single-labeled probes.