The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes

The efficiency of DNA labeling was assessed for 2'-deoxyuridine 5'-triphosphate (dUTP) derivatives containing the Cy7 cyanine dye as a fluorophore. Two fluorescent Cy7-labeled dUTP analogs differed in the chemical structure of the linker between the fluorophore and nucleotide moieties. The efficiency of the polymerase chain reaction (PCR) and inhibition with modified nucleotides were estimated by real-time PCR. The efficiency of labeled nucleotide incorporation in PCR products was measured by quantitative electrophoresis. The efficiency of target DNA labeling was evaluated by binding the fluorescently labeled PCR products to a microarray of oligonucleotide probes immobilized in hydrogel drops (a biochip). The near-infrared hybridization signal was detected by digital luminescence microscopy. An increase in linker length was found to provide more efficient incorporation of the labeled nucleotide. Both of the compounds provided high sensitivity and high specificity of DNA testing via allele-specific hybridization on a biochip.

     

PrimRglo: a multiplexable quantitative real-time polymerase chain reaction system for nucleic acid detection

We report the development of a new real-time polymerase chain reaction (PCR) detection system that uses oligonucleotide "tagged" PCR primers, a fluorophore-labeled "universal" detection oligonucleotides, and a complementary quenching oligonucleotide. The fluorescence signal decreases as PCR product accumulates due to the increase in detection/quencher hybrid formation as the tagged primer is consumed. We use plasmids containing the influenza A matrix gene and the porA and ctrA genes of Neisseria meningitidis as targets for developing the system. Cycle threshold (Ct) values were generated, and the sensitivity of the new system (dubbed "PrimRglo") compared favorably with the commonly used SYBR green and Taqman detection systems and, unlike the latter system, does not require the design of a new dual-labeled detection oligonucleotide for each new target sequence.     

Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator.     

Synthesis and properties of fluorescent NF-kappa B-recognizing hairpin oligodeoxyribonucleotide decoys

Intramolecular fluorescence quenching of cyanine dyes was investigated using a model hairpin oligonucleotide decoy encoding a NF-kappaB p50 subunit binding site. Two types of hairpin oligonucleotides were synthesized: (1) 5'-(6-aminohexyl)- and 3'-(3-aminopropyl)-linked (I); (2) 5'-(6-aminohexyl)- and 3'-[3-(3-hydroxypropyldithio)propyl]-linked (II). Oligonucleotide I was covalently modified using monofunctional either Cy3- or Cy5.5-N-hydroxysuccinimide esters. Using reverse-phase HPLC, mono-and dicyanineamide derivatives of I were isolated. Mono-Cy3-modified derivatives of I, but not the mono-Cy5.5-modified derivatives, showed a 2-fold higher Cy3 fluorescence intensity compared to the free dye. There was no detectable difference in fluorescence between the di-Cy3 derivative of I and the free dye at the same concentration. However, there was a 4-fold quenching of fluorescence in the case of the di-Cy5.5 derivative of the same hairpin oligonucleotide. The quenching of Cy5.5 fluorescence could not be explained by the interaction of Cy5.5 with nucleotide bases as demonstrated by incubating free Cy5.5 dye with oligonuclotides. The quenching effect was further investigated using an oligonucleotide bearing a cleavable 3'-amino-terminated linker bearing an S-S bond (III). After modification of the 5'- and 3'-end of oligonucleotide III with a Cy5.5 monofunctional hydroxysuccinimide ester, a 70-75% quenching of fluorescence was observed. Fluorescence was 100% dequenched after the reduction of S-S bond. The obtained result unequivocally demonstrates that the formation of intramolecular Cy5.5 dimers is the dominant mechanism of fluorescence quenching in symmetric dye-dye hairpin decoy beacons.     

Array-based mutation detection of BRCA1 using direct probe/target hybridization

We describe here an efficient microarray-based multiplex assay to detect Korean-specific mutations in breast cancer susceptibility gene BRCA1 using direct probe/target hybridization. Allele-specific oligonucleotides were covalently immobilized on an aldehyde-activated glass slide to prepare an oligonucleotide chip. From a wild-type sample, a two-step method was used to generate labeled multiplex polymerase chain reaction (PCR) amplification products of genomic regions containing the mutation sites. Amino allyl-dUTP, an amine-modified nucleotide, was incorporated during multiplex PCR amplifications and a monofunctional form of cyanine 3 dye was subsequently attached to the reactive amine group of the PCR products. Hybridization of the labeled PCR products to the oligonucleotide chip successfully identified all of the genotypes for the selected mutation sites. This work demonstrates that oligonucleotides chip-based analysis is a good candidate for efficient clinical testing for BRCA1 mutations when combined with the indirect strategy to prepare labeled target samples.     

实时荧光定量PCR在固氮酶基因检测中的应用

实时荧光定量PCR是近年发展起来的一种新的实时定量检测特定核酸技术,它是核酸探针技术、荧光共振能量传递技术和PCR技术的有机结合。与常规PCR相比,它具有特异性更强、能有效解决PCR污染问题、自动化程度高等特点,扩大了PCR的应用范围。概述实时荧光定量PCR技术在固氮酶(nifH)基因检测中的应用与研究进展,并探讨该技术的发展和应用前景。     

The Effect of the Structure of Fluorescently Labeled Nucleotide Derivatives on the Efficiency of Their Incorporation in DNA in the Polymerase Chain Reaction

The efficiency of fluorescence DNA labeling was estimated for four fluorescent 2′-deoxyuridine 5′-triphosphate derivatives differing in the orientation of the main dye axis, which passes through the polymethine chain, relative to the linker connecting the dye to the nucleotide. To estimate the polymerase chain reaction (PCR) rate, real-time PCR was run with two commercial hot-start DNA polymerases possessing 5′→3′ exonuclease activity in the presence of an intercalating dye. The efficiency of the test compound incorporation in the PCR product was estimated via a quantitative analysis of the amplification product by agarose gel electrophoresis. The fluorescently labeled product was then hybridized on a biological microchip and the ratio of signals from perfect match and mismatch duplexes was determined. The incorporation efficiency and discrimination between perfect match and mismatch duplexes were found to depend on the relative orientation of the dye and the linker between the dye and pyrimidine base, as well as on the presence of hydrophilic groups in the dye. Compounds that are efficiently incorporated in a growing DNA strand and show a high specificity in hybridization analysis were identified using biochips.     

A new minor groove binding asymmetric cyanine reporter dye for real-time PCR

The minor groove binding asymmetric cyanine dye 4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene)]-1-methyl-pyridin ium iodide (BEBO) is tested as sequence non- specific label in real-time PCR. The fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations between 0.1 and 0.4 µM generated sufficient fluorescence signal without inhibiting the PCR. A comparison with the commonly used reporter dye SYBR Green I shows that the two dyes behave similarly in all important aspects.     

Anti-primer quenching-based real-time PCR for simplex or multiplex DNA quantification and single-nucleotide polymorphism genotyping

Nucleic acid amplification and detection plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics and drug discovery. We present a new quantitative PCR method that allows versatile and flexible nucleic acid target quantification. One of the PCR primers is modified by an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3'-quencher ("anti-primer"), is included in the PCR along with the two primers. Following primer extension, the reaction temperature is lowered such that the anti-primer hybridizes to and quenches the fluorescence of only the free primer and not the double-stranded PCR product, allowing real-time fluorescent quantification of the latter. This anti-primer-based quantitative real-time PCR (aQRT-PCR) allows simplex or multiplex quantification or single-nucleotide polymorphism genotyping in clinical samples of widely differing quality (e.g., fresh samples, formalin-fixed paraffin-embedded samples and plasma-circulating DNA) and provides a practical alternative to existing, more expensive approaches. The process of aQRT-PCR takes 1.5-2 h.     

Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10°C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.     

Detection of the Hereditary Hemochromatosis Gene Mutation by Real-Time Fluorescence Polymerase Chain Reaction and Peptide Nucleic Acid Clamping

Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new double-stranded DNA-selective fluorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that fluorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.     

Interaction of cyanine dyes with nucleic acids. Part 19: new method for the covalent labeling of oligonucleotides with pyrylium cyanine dyes

New chemistry for the fluorescent labeling of oligonucleotides with cyanine dyes is proposed. It is based on the use of pyrylium salts as amine-specific reagents. Monomethyne pyrylium cyanine dye 1 was covalently linked to 5'-aminoalkyl modified oligonucleotide, with simultaneous conversion of the non-fluorescent dye 1 into fluorescent pyridinium cyanine structure 2.     

Rapid screening of libraries with radiolabeled DNA sequences generated by PCR using highly degenerate oligonucleotide mixtures.

The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.     

The kinetics of fluorescent DNA labeling using PCR with different Taq polymerases depends on the chemical structures of modified nucleotides

The kinetics of DNA labeling during PCR using six fluorescent derivatives of 2′-deoxyuridine 5′-triphosphate has been studied. These compounds differ in their chemical structure, total electric charge and the length of the linker between a dye and the C5 position of a pyrimidine base. The efficiency of the incorporation of the fluorescent derivatives into a growing DNA chain by four commercially available Taq DNA polymerases with 5′→3′ exonuclease and hot start activity has been determined using real-time PCR with a TaqMan probe and the subsequent electrophoretic analysis of the reaction products. Modified deoxyuridines with a total positive or negative charge of the chromophore were practically not incorporated by Taq polymerases during PCR. The modified deoxyuridines with a neutral charge of the chromophore were effectively incorporated into DNA. The extended length of the linker between the pyrimidine base and the chromophore led to a lower PCR inhibition and a more effective inclusion of modified nucleotides in the growing DNA chain. This fact can be explained by the reduced steric effects that were caused by the dye. As a result, the most promising combinations of fluorescently labeled nucleotide and Taq polymerase have been chosen for further use in fluorescent DNA labeling.     

Loop-mediated isothermal amplification for the detection of goose circovirus

ABSTRACT: BACKGROUND: Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. Theinfection's clinical symptoms include growth retardation or feathering disorders but theinfection process may remain non-symptomatic what makes the infected birds moresusceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection ismade by histopathological examination, dot blot hybridization, polymerase chain reaction(PCR) and real-time PCR. However these techniques require application of thermocyclersand qualified staff which may be cost-consuming for some diagnostic units. The aim of thisstudy was to develop loop-mediated isothermal amplification assay (LAMP) as a simplemethod of GCV detection. RESULTS: The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus(GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate thereaction. The optimum reaction temperature and the time were 62°C for 30 min, respectively.The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eightisolates of GCV collected from geese flocks in Poland were examined. For comparison, realtimepolymerase chain reaction with F3 and B3 primers and SYBR Green dye wasconducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specificassay and alternative for PCR-based methods. CONCLUSIONS: The developed technique due to its simplicity may be applied by any veterinary laboratory oreven mobile diagnostics units for the routine detection of GCV.     

Reliability of detecting rRNA sequences of Chlamydia trachomatis with fluorescence in situ hybridization without amplification

OBJECTIVE: To use fluorescence in situ hybridization (FISH) using ribosomal RNA (rRNA) oligonucleotide probes as the target nucleic acid for the detection of Chlamydia trachomatis. STUDY DESIGN: Suitable sequences selected from the rRNA sequence of C trachomatis were labeled with a fluorescent dye and used in FISH for detecting chlamydial inclusion bodies and/ or elementary bodies in paraformaldehyde-fixed urogenital swab samples. The sensitivity and specificity of the FISH assay were compared with those of the polymerase chain reaction (PCR) using plasmid primers. Positive known C trachomatis-infected McCoy cells were used as positive controls. Urogenital swab specimens that were C trachomatis negative on culture and PCR were used as negative controls. RESULT: Among the 128 samples included in the study, FISH was positive in 28 (21.8%) and PCR in 33 (25.7%). A significant correlation was found between the 2 detection methods. Results of PCR and FISH were consistent in 115 of the 128 samples (R = 0.89). Thirteen samples showed discordant results. Of these, 9 FISH negative samples were PCR positive and 4 FISH positive samples were PCR negative. CONCLUSION: FISH was a highly specific and fairly sensitive technique for detecting C trachomatis. Signal amplification techniques and use of different fluorophores may further increase the sensitivity of this technique.     

The enhancement of PCR amplification by low molecular-weight sulfones.

DNA amplification by polymerase chain reaction (PCR) is frequently complicated by the problems of low yield and specificity, especially when the GC content of the target sequence is high. A common approach to the optimization of such reactions is the addition of small quantities of certain organic chemicals, such as dimethylsulfoxide (DMSO), betaine, polyethylene glycol and formamide, to the reaction mixture. Even in the presence of such additives, however, the amplification of GC-rich templates is often ineffective. In this paper, we introduce a novel class of PCR-enhancing compounds, the low molecular-weight sulfones, that are effective in the optimization of high GC template amplification. We describe here the results of an extensive structure-activity investigation in which we studied the effects of a series of six different sulfones on PCR amplification. We identify two sulfones, sulfolane and methyl sulfone, that are especially potent enhancers of high GC template amplification, and show that these compounds often outperform DMSO and betaine, two of the most effective PCR enhancers currently used. We conclude with a brief discussion of the role that the sulfone functional group may play in such enhancement.     

Synthesis of Unsymmetrical Polymethine Cyanine Fluorescent Dyes for Nucleic Acid Analysis by Real-Time PCR

The synthesis of unsymmetrical polymethine cyanine derivatives of f luorescent dyes is described, and their potential as a component of hybridization probes for the real-time PCR technique is first demonstrated.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.