Simple detection of point mutations in DNA oligonucleotides using SYBR Green I

A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (intercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 pmol of sample DNA.     

基于SYBR Green I的双链DNA定量方法

摘要 基于SYBR Green I荧光染料与双链DNA（dsDNA）结合产生荧光的原理,建立一种高精度、高通量的双链DNA 定量方法。将梯度稀释后的基因组DNA及已知浓度的?DNA与等体积的SYBR Green I（4×）充分混合后,利用荧光定量PCR仪采集荧光信号,以ROX（1×）作为校正染料进行定量分析;同时利用紫外分光光度计对样品进行平行测定,比较该方法与紫外分光光度法的检测限与准确度。紫外分光光度法的检测限为2 ng/?l,而SYBR Green I荧光定量法的检测限可达到0.015 ng/?l,并且在0.015~2 ng/?l范围内,SYBR Green I荧光强度与?DNA浓度呈线性关系（R2=0.9999）,比紫外分光光度法灵敏100倍以上,并可准确定量低纯度的DNA样品。此方法具有重复性好、高通量的特点,仅需少量的生物样本即可满足定量要求,为分子生物学研究及临床检验等多个领域提供了一种可靠的dsDNA定量方法。     

Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye

A high-throughput, 96-well microplate fluorescence assay (MFA) was developed for DNA quantification using the double-stranded DNA-binding dye SYBR Green I. Samples mixed with SYBR Green I in the wells of a microtiter plate produced fluorescence in proportion with DNA concentration which was measured using a fluorescence plate reader. The performance characteristics of the assay were compared with spectrophotometric quantification based on ultraviolet absorption and the Hoefer DyNA Quant assay utilizing the fluorescent dye, Hoechst 33258. The MFA accurately quantified different types of DNA over a broad linear dynamic range of concentrations (0.25–2,500 pg/μl), and was not affected by a variety of contaminants in the assay mixture.     

Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T_m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T_m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T_m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T_m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.     

Quantitative detection of Proteus species by real-time polymerase chain reaction using SYBR Green I

A SYBR Green real-time polymerase chain reaction (PCR) method for rapid detection of Proteus species was developed and evaluated. Of 322 clinical and food samples tested, 75 samples were positive for Proteus species by using conventional PCR and real-time PCR assays. The results were consistent with standard culture methods and the Vitek auto-microbe system, indicating a 100 % specificity obtained by both PCR assays. For the real-time PCR method, the minimum detectable level was 10 colony forming units (CFU) /ml, which was a 10³ multiple higher than the conventional PCR method. Correlation coefficients of standard curves which were constructed using the threshold cycle (Ct) versus copy numbers of Proteus showed good linearity (R ²?=?0.997). In conclusion, several significant advantages such as higher sensitivity and rapidness were observed by using the SYBR Green real-time PCR method for identifying Proteus species.     

A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system

Various assays are available for quantification of DNA in solution, but none has been described that is both sensitive and specific for double-stranded (ds) DNA and features practical properties such as low dye and equipment costs, speed, and highly parallel microplate formats. Here we show that quantitative and sensitive measurement of ds DNA in solution is achieved using a 96-well microplate SYBR Green I assay and a standard uv transillumination-based gel-imaging system for detection. Specific detection of ds DNA was obtained over a broad concentration range of 0.5-500 ng using a single low dye concentration of up to 1/6250. Measured SYBR Green I fluorescence was not significantly affected by pH variation (4-10), assay volume (50-250 microliter l), and time (4-15 min), and measurements were appreciably compatile with commonly encountered concentrations of contaminating salts, organics, detergents, and other substances. ds DNA yielded up to 13-fold higher fluorescence compared to single-stranded DNA or RNA, but this ratio was dependent somewhat on GC content and fragment size. Of note, linear ds DNA fluoresced significantly stronger than supercoiled plasmid DNA. Our method should be broadly applicable for sensitive, rapid, and inexpensive ds DNA quantification in the average molecular biology laboratory.     

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure–property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above ~0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA) · poly(dT) and poly(dG) · poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least ~11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]⁺. These structure–property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.     

Driving proteins off DNA using applied tension.

Proteins that bind DNA so as to reduce its end-to-end length can be dissociated by application of force. The thermodynamics of this process are discussed, with special attention to the case of histones bound to DNA (i.e., a string of nucleosomes, or chromatin fiber). The histone octamer is predicted to be driven off chromatin fiber for tensions >2 piconewtons.     

SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This method is as sensitive as [3H]thymidine prelabelling of cells and allows DNA dsb to be measured simply and economically in non-cycling cells.     

A real-time PCR assay for the detection and quantitation of Campylobacter jejuni using SYBR Green I and the LightCycler

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional biochemical identification for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable (VNC) state. Nucleic acid-based tests have emerged as a useful alternative to traditional testing. In this article, we present fluorescent quantitative PCR assay for quantitative detection of C. jejuni, the assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double-stranded DNA binding dye SYBR Green I. When this assay was applied, the assay positive for all of the isolates of C. jejuni tested (11 isolates, including type strain ATCC33560) and negative for all other Campylobacter spp. (three isolates) and several other bacteria (five species tested). The total assay could be completed in 60 min with a detection limit of approximately 1 CFU, and a correlation coefficient was 1.000. Result indicated that fluorescent quantitative detection methods provided a special, sensitive, rapid, reproducible and accurate method for quantitative detection of C. jejuni.     

Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems

In an in-gel polymerase chain reaction (PCR), the generation of a 1750-bp yeast DNA fragment was inhibited when yeast DNA gel-stabs or gel-slices stained with ethidium bromide (EtBr) or SYBR Green I were used. Similar inhibition occurred to a varying degree in the reamplification of PCR fragments in prokaryotic systems. Inclusion of the dyes in PCR resulted in an inhibition at about 10 microg/ml EtBr and at 10,000-20,000-fold dilution of SYBR Green I in all systems. The effect remained unchanged despite increasing the PCR cycles to 40. However, increasing the magnesium chloride concentration did reverse the inhibitory actions, although the PCR specificity was lost. In an unusual observation, we find that, at higher dye concentrations (50 microg/ml EtBr, or thousand fold dilution of SYBR Green I), the input yeast DNA electrophoretic profile is maintained following 25 PCR cycles (despite a denaturation temperature of 94 degrees C). It varied significantly in different DNA systems and was readily reversed by high Mg++ concentrations. It is concluded that, at low Mg++ concentrations, different PCR systems are inhibited to varying extents by intercalating dyes and, in some PCR systems, intercalating dyes at unusually high concentrations maintain input DNA electrophoretic profile.     

Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli

Ethidium bromide (EtBr) and SYBR Green I are nucleic acid gel stains and used commonly in combination with UV-illumination. EtBr preferentially induces frameshift mutations but only in the presence of an exogenous metabolic activation system, while SYBR Green I is a very weak mutagen that induces frameshift mutations. We found that EtBr and SYBR Green I, without an added metabolic activation system, strongly potentiated the base-substitution mutations induced by UV-irradiation in E. coli B/r WP2 cells. Each DNA stain alone showed no mutagenicity to the strain. Base-substitutions induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and 4-nitroquinoline-1-oxide were similarly potentiated by EtBr and SYBR Green I. SYBR Green I had a much greater effect. No enhancing effects were observed on mutations induced by mitomycin C, cisplatin, transplatin, cumene hydroperoxide, base analogs, and alkylating agents. Another DNA stain, acridine orange, showed similar enhancing effects on UV- and MX-mutagenicity, but no effect was observed for 4',6-diamidino-2-phenylindole (DAPI). UV- and MX-induced mutations in E. coli WP2s (uvrA), which is defective in nucleotide excision repair activity, were not potentiated by the addition of EtBr, SYBR Green I, or acridine orange. Those nucleic acid stains might inhibit the nucleotide excision repair of DNA damaged by UV and MX treatment.     

Using fluorescent nanoparticles and SYBR Green I based two-color flow cytometry to determine Mycobacterium tuberculosis avoiding false positives

A method using an improved two-color flow cytometric analysis by a combination of bioconjugated fluorescent silica nanoparticles and SYBR Green I (FSiNP@SG-FCM) has been developed for detection of pathogenic Mycobacterium tuberculosis. Antibody-conjugated nanoparticles were prepared by oriented immobilization of the anti-M. tuberculosis antibody onto Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate-doped fluorescent silica nanoparticles (RuBpy-doped FSiNPs) through Protein A. M. tuberculosis was specially labeled with antibody-conjugated RuBpy-doped FSiNPs, then stained with a nucleic acid dye SYBR Green I to exclude background detrital particles, followed by multiparameter determination with flow cytometry. With this method, false positives caused by aggregates of nanoparticle-bioconjugates and nonspecific binding of nanoparticle-bioconjugates to background debris could be significantly decreased. This assay allowed for detection of as low as 3.5 x 10(3) and 3.0 x 10(4)cellsml(-1)M. tuberculosis in buffer and spiked urine respectively, with higher sensitivities than the FITC-based conventional flow cytometry. The total assay time including sample pretreatment was within 2h. This proposed FSiNP@SG-FCM method will be promising for rapid detection of M. tuberculosis or other pathogenic bacteria in clinical samples.     

A new model for DNA containing A.T and I.C base pairs.

DNA polymers containing exclusively A.T or I.C base pairs frequently exhibit D- or E-type X-ray diffraction patterns when dried. The distribution of intensities in fiber patterns appears to demand helical structures with 7 and 7.5 bp/turn, respectively, but it is not stereochemically possible to wind a right-handed antiparallel B-family helix this tightly. It is a simple matter, however, to build a left-handed helix with 7-7.5 bp/turn by incorporating Hoogsteen pairing into a Z helix framework. X-ray intensities calculated from this novel left-handed Hoogsteen model provide as reasonable a fit to the D-DNA diffraction pattern as do intensities calculated from previously proposed right-handed 8-fold models.     

Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T_m) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T_m, which was consistently specific for the amplicon obtained; the mean peak T_m obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10³ to 10⁸ CFU/ml) showed good linearity (R² = 0.9767) and a sensitivity limit of less than 10³ CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.     

SYBR green实时荧光定量PCR检测微小RNA21的方法建立及在食管鳞癌中的应用

目的:建立SYBR green实时荧光定量PCR检测微小RNA miR-21的技术平台及应用。方法:设计微小RNA21和U6的的颈环结构反转录引物和PCR扩增引物,以U6为内参利用SYBR green实时荧光定量PCR法检测小鼠各器官中的微小RNA21的含量。提取16例食管鳞癌患者的肿瘤组织及其近旁组织中的总RNA,检测其微小RNA21表达水平。结果:SYBR green实时荧光定量PCR检测U6和微小RNA21含量的熔解曲线单一,PCR产物特异。在Balb/c小鼠的4种器官中,肝脏、脾脏、肾脏分别为脑组织的8.71、5.38、3.47倍。16对食管鳞癌患者的样本中,14例微小RNA21的拷贝数高于其近旁组织约10.58倍(p0.01)。结论:此研究成功建立了SYBR green荧光定量PCR法检测小鼠和人微小RNA-21含量的技术平台,为进一步阐述miR-21在食管鳞癌的发生中的作用提供了新方向。     

Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II

A simple, non-destructive procedure is described to determine the quality of DNA arrays before they are used. It consists of a preliminary staining step of the DNA microarray by using SYBR green II, a fluorophore with specific affinity for ssDNA, followed by a laser scan analysis. The surface quality, integrity and homogeneity of each DNA spot of the array can thus be assessed. After this preliminary control, which may avoid further analytical steps that lead to the waste of precious biological samples, a fully reversible staining procedure is performed that produces an array ready for subsequent use.