Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.     

High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer

In the initial report, introducing a single phosphorothioate modification at the very 3' terminus of the oligodeoxynucleotide primer has been shown to effectively protect the oligodeoxynucleotide degradation due to the 3' exonuclease activity. In this study, we reported a novel finding that phosphorothioate modification at the 3' end of primers could not only effectively prevent the primer from degradation, but could also mediate an off-switch extension by Pfu polymerase when primers also carry single or multiple mismatched bases located in the first eight bases of the 3' terminus. This suggests that the combination of 3' phosphorothioate-modified primers with exo+ polymerases such as Pfu constituted an on/off switch, which allows perfectly matched primers to be extended but not mismatched primers. Furthermore, we found that polymerases with different fidelities showed different efficiencies in turning off mismatched-primer mediated extension. So we described here a SYBR green-based real-time quantitative PCR assay for the detection of abundance level of gene expression that did not require fluorescently labeled gene-specific probes or complicated primer combinations. The emergence of real-time quantitative RT-PCR technology is thus suited for a diverse application with a need for high-throughput methods to detect and quantify different gene expressions by way of simplicity, versatility, and accuracy, and thus could complement global microarray-based expression profiling strategies.     

Monomeric red fluorescent protein as a reporter for macromolecular localization in Streptomyces coelicolor

The enhanced green fluorescent protein (eGFP) is widely used to investigate cell type specific gene expression and protein localization in the filamentous streptomycetes. To broaden the scope of cell biological investigation in these organisms, we have adapted shuttle vectors for the construction of gene fusions to the monomeric red fluorescent protein (mRFP1) and have tested them in Streptomyces coelicolor. Using fusions of mRFP1 to the cell division proteins DivIVA and FtsZ, we show that mRFP1 is comparable to eGFP for cell biological research in this organism and suggest that this paves the way for the future use of two-color imaging and FRET.     

Analysis of expression profiles of three peroxidase genes associated with lignification in Arabidopsis thaliana

We have investigated the mechanism of lignification during tracheary element (TE) differentiation using a Zinnia elegans xylogenic culture. In the process, we isolated ZPO-C , a peroxidase gene of Z. elegans that is expressed specifically in differentiating TEs. ZPO-C is suggested to be involved in lignification of Z. elegans TEs in vivo and in vitro. Furthermore, a peroxidase gene of Arabidopsis thaliana ( AtPrx66 ), which is homologous to ZPO-C , was identified. The expression profile and functions of the gene in planta remain to be investigated. In this study, we performed promoter :: β-glucuronidase (GUS) assays to investigate the expression profiles and functions of the ZPO-C -like peroxidases in A. thaliana . We generated transgenic A. thaliana lines carrying AtPrx66, AtPrx47 or AtPrx64 (peroxidases showing high sequence similarity to AtPrx66 ) promoter :: GUS reporter gene fusions. The GUS activities of AtPrx66, AtPrx47 and AtPrx64 promoter :: GUS lines were arranged concentrically from the center to the periphery in the roots of seedlings. Furthermore, histochemical GUS assays using inflorescence stems showed that AtPrx66, AtPrx47 and AtPrx64 promoter-driven GUS were mainly expressed in the differentiating vessels, xylem parenchyma and sclerenchyma, respectively. These results suggest that the gene expressions of these three peroxidases, which showed high sequence similarity to one another, are differentially regulated in various tissues and organs. In addition, our results suggest that while AtPrx66 and AtPrx47 are associated with lignification of vessels, AtPrx64 is associated with lignification of sclerenchyma.     

The synthesis of oligonucleotides containing an aliphatic amino group at the 5'' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis.

A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to synthesize several fluorescent derivatives of an oligonucleotide primer used in DNA sequence analysis by the dideoxy (enzymatic) method. The modified primers are highly fluorescent and retain their ability to specifically prime DNA synthesis. The use of these fluorescent primers in DNA sequence analysis will enable DNA sequence analysis to be automated.     

Universal fluorescent labeling (UFL) method for automated microsatellite analysis.

We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.     

毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

Global analysis of gene expression by differential display

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. However, there has been lack of an accurate guidance on how many DD polymerase chain reaction (PCR) primer combinations are needed to display most of the genes expressed in a eukaryotic cell. This study critically evaluated the gene coverage by DD as a function of the number of arbitrary primers, the number of 3′ bases of an arbitrary primer required to completely match an mRNA target sequence, the additional 5′ base match(s) of arbitrary primers in first-strand cDNA recognition, and the length of mRNA tails being analyzed. The resulting new DD mathematical model predicts that 80 to 160 arbitrary 13mers, when used in combinations with 3 one-base anchored oligo-dT primers, would allow any given mRNA within a eukaryotic cell to be detected with a 74% to 93% probability, respectively. The prediction was supported by both computer simulation of the DD process and experimental data from a comprehensive fluorescent DD screening for target genes of tumor-suppressor p53. Thus, this work provides a theoretical foundation upon which global analysis of gene expression by DD can be pursued.     

Rapid identification of single nucleotide polymorphisms by fluorescence-based capillary electrophoresis

We describe the application of two different fluorescence-based techniques (ddNTP primer extension and single-strand conformation polymorphism (SSCP)) to the detection of single nucleotide polymorphisms (SNPs) by capillary electrophoresis. The ddNTP primer extension technique is based on the extension, in the presence of fluorescence-labeled dideoxy nucleotides (ddNTP, terminators), of an unlabeled oligonucleotide primer that binds to the complementary template immediately adjacent to the mutant nucleotide position. Given that there are no unlabeled dNTPs, a single ddNTP is added to its 3' end, resulting in a fluorescence-labeled primer extension product which is readily separated by capillary electrophoresis. On the other hand, the non-radioisotopic version of SSCP established in this study uses fluorescent dye to label the PCR products, which are also analyzed by capillary electrophoresis. These procedures were used to identify a well-defined SNP in exon 7 of the human p53 gene in DNA samples isolated from two human cell lines (CEM and THP-1 cells). The results revealed a heterozygous single-base transition (G to A) at nucleotide position 14071 in CEM cells, proving that both fluorescence-based ddNTP primer extension and SSCP are rapid, simple, robust, specific and with no ambiguity in interpretation for the detection of well-defined SNPs.     

In situ amplification using universal energy transfer-labeled primers.

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used.     

Homogeneous real-time detection and quantification of nucleic acid amplification using restriction enzyme digestion

A method for real-time fluorescent detection and quantification of nucleic acid amplification using a restriction endonuclease was developed. In this homogeneous system detection is mediated by a primer containing a reporter and quencher moiety at its 5' terminus separated by a short section of DNA encoding a restriction enzyme recognition sequence. In the single stranded form, the signal from the fluorescent reporter is quenched due to fluorescence resonance energy transfer. However, as the primer becomes incorporated into a double stranded amplicon, a restriction enzyme present in the reaction cleaves the DNA linking the reporter and quencher, allowing unrestricted fluorescence of the reporter. To test this system, a primer specific for the E6 gene of human papilloma virus (HPV) 16 was combined with the cleavable energy transfer label and used to amplify HPV16 positive DNA. In the presence of the thermally stable restriction enzyme BstNI, the reporter system was found to generate a fluorescent signal in proportion to the amount of template DNA. In addition to this direct format, the reporter primer was also used to monitor and quantify the amplification of other sequences. This was accomplished by using primers that contain a tag sequence complementary to the reporter oligonucleotide.     

Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.     

Improved biplex quantitative real-time polymerase chain reaction with modified primers for gene expression analysis

Stabilizing modified bases incorporated in primers allows the reduction of housekeeping gene primer concentration not possible with regular primers without sacrificing amplification efficiency. Low primer concentration allows coamplification of the most abundant housekeeping genes with very rare templates without mutual inhibition. Real-time polymerase chain reaction (PCR) coamplification of 18S ribosomal RNA with several genes of interest was used in this study with MGB Eclipse (Nanogen, San Diego, CA) hybridization probes. The results may be useful for high throughput gene expression studies as they simplify validation experiments.     

A universal primer mixture for sequence determination at the 3' ends of cDNAs

We have devised a universal primer which can be used to sequence the 3'-ends of cloned cDNAs containing a polyA tail. The primer consists of an equimolar mixture of three primers: 20 T nucleotides followed by either an A, C, or G nucleotide (5'----3'). With this primer mixture and the dideoxynucleotide chain termination method, we determined the 3'-terminal sequence of human beta-actin cDNA in an Okayama-Berg vector, in four parallel sets of reactions containing either a single primer (T20G, T20C, or T20A) or an equimolar mixture of all three primers. Priming with both T20A and the triple mixture gave clearly readable results that agree with the known sequence of the human beta-actin gene, and we have applied this method successfully to several other cDNAs in the Okayama-Berg expression vector. Use of this universal primer mixture facilitates determination of sequences at the 3'-ends of cDNAs while by-passing the polyA tail region.