Capillary electropherograms for restriction fragment length polymorphism of Helicobacter pylori

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.     

Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis

The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser‐induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE‐LIF‐REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE‐LIF. The results demonstrate that the CE‐LIF‐REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.     

Genetic evidence of an Asian background in heteroplasmic Iberian cattle (Bos taurus): effect on food authentication studies based on polymerase chain reaction-restriction fragment length polymorphism analysis

This work was aimed at identifying nucleotide polymorphic sites in a 359 bp region of the cytochrome b (cytb) mitochondrial gene of Iberian cattle (Bos taurus). This region is widely used as target in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) species identification studies in foodstuffs destined for human and animal consumption. Two different coexisting restriction patterns were observed in four of the six animals studied when the 359 bp DNA fragment was cleaved with PalI, HinfI, MvaI, RsaI, or MboI. The amplification of both genotypes with the mitochondrial-specific primers L14735 and H15149 revealed the absence of nuclear pseudo-cytb genes, confirming the existence of mitochondrial heteroplasmy. The two coexisting mtDNA fragments were selectively sequenced in PCR extracts in which one genotype predominated over the other, both exhibiting a sequence variation of 10.4%. From the 37 nucleotide mismatches observed between genotypes, 32 were transitions and five were transversions. While 31 of the nucleotide mismatches between genotypes resulted to be conservative at the amino acid level, six changes implied amino acid substitutions, five of them being located in the variable transmembrane region. Genetic analysis suggests the presence of an Asian background in the mitochondria of Iberian cattle: while one of the genotypes matched the published sequence for Bos taurus, the other genotype clustered with a B. primigenius indicus animal and close to an Asian Bos taurus animal. These results also suggest that a number of current PCR-RFLP species identification methods based on cytb sequences may not be reliable for the accurate detection and identification of bovine material: an alternative battery of enzymes consisting of MmeI, NlaIV, and AluI is proposed.     

Improved DNA fragment length estimation in capillary electrophoresis

In CE the charged DNA strands are fractionated according to fragment lengths as they migrate through the capillary, since shorter DNA fragments pass through the sieving matrix faster. Multiplexed internal size standards are used to estimate the size of unknown DNA fragments. In the literature there are statements about migration abnormalities for the 250 and 340 bp fragments in the GeneScan-500 (GS500) internal size standards. Such anomalous migration of size standards could obviously introduce errors in the estimation of unknown fragments. Therefore, a number of analysis programs simply exclude some of these fragments. In the present work we first evaluate the effect of excluding each of the fragments in the internal size standards used in CE. Next, a method which is based on estimating the true values of the anomalous fragments is presented. The results obtained by the new method indicate a significant improvement compared to results obtained when one (or both) of the anomalous fragments in GS500 is (are) excluded or included when estimating the size of unknown DNA fragments. In the higher-molecular-weight region, the average error is reduced from 1.91 bp in ABI GeneMapper (excluding 250 bp) to 0.15 bp in the new method (using the estimated values for 250 and 340 bp). In the lower-molecular-weight region, excluding both fragments will improve the results by an average of 0.74 bp compared to ABI GeneMapper.     

Genotyping and haplotyping of the dopamine D4 receptor gene by capillary electrophoresis

In this paper we report on simultaneous genotyping of adjacent polymorphisms (referred to as haplotyping) by combining double-tube allele-specific polymerase chain reaction, restriction fragment length polymorphism and capillary gel electrophoresis analysis of the resulting fragments. Direct molecular haplotyping is of particular importance in the case of double heterozygote samples, since in these instances the haplotype structure cannot be constructed based on genotype data. Our approach provided a powerful tool for coincidental genotype analysis of the 48 base pair (bp) variable number of tandem repeats of the third exon and haplotype investigation of the -616CG and -521CT single nucleotide polymorphisms of the dopamine D4 receptor (DRD4) gene. The linear polyacrylamide sieving matrix was optimized for the size range of the double-stranded DNA fragments of interest varying from 35 to 763 bp. We demonstrated that capillary gel electrophoresis in combination with laser induced fluorescence detection offers a sensitive and accurate tool for automated haplotyping in clinical settings.     

样品稀释对毛细管电泳分离检测DNA片段的影响

通过理论推导和实验验证表明;适当稀释DNA样品溶液,采用流体力学进样或电动进样都不会较大地减低峰高,而DNA片段毛细管电泳的分离效率和分离度还能有所提高。采用稀释样品的方法可提高DNA样品的使用效率。采用羟乙基纤维素无胶筛分介质分离了DNA片段。用激光诱导荧光（氩离子激光器,488nm）电荷耦合器件检测。用低浓度的筛分介质（0．4％）分离了分子质量较大的ADNA－HindⅢ全部8个片段（12bp～23130bP）。用高浓度的筛分介质（1．6％）分离分子质量较小的pBR322－HaeⅢ22个片段（18bp～587bp）。     

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.     

Monodisperse DNA restriction fragments I. Synthesis and characterization

We present a convenient and low-cost method to prepare milligram amounts of completely monodisperse DNA restriction fragments in a physico-chemical laboratory setting to study (in part II) the effect of limited flexibility on the concentration dependent sedimentation velocity. Four fragments of 200, 400, 800, and 1600 bp were designed to span a range of 1-11 persistence lengths. The fragments were synthesized by cloning fragments of controlled lengths obtained by PCR into bacterial plasmid DNA. The constructs were amplified in large-scale bacterial cultures from which the fragments were obtained by a modified alkaline lysis procedure and subsequent digestion with EcoRV. A method is presented to isolate the DNA from the digestion mixture using horizontal agarose-slab gels and agarose columns in a home-built preparative gel electrophoresis set-up. We show that a combination of optical absorbance readings, ethidium bromide fluorescence, and hyperchromicity measurements allows assessment of both the purity of the DNA solutions and the fraction of double-stranded DNA.     

A study of comparability in amplified fragment length polymorphism profiling using a simple model system

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.     

毛细管电泳-激光诱导荧光检测分枝杆菌脱氧核糖核酸限制性内切酶谱

建立了毛细管电泳分离-激光诱导荧光检测（CE-LIFD）分析分枝杆菌脱氧核糖核酸（DNA）限制性内切酶谱的新方法。用聚合酶链反应(PCR)扩增分枝杆菌hsp65基因的长度为439 bp的片段,该扩增片段经限制性内切酶BstEⅡ和 HaeⅢ酶切后,分别用CE-LIFD装置和常规琼脂糖电泳（AGE）对比检测酶切片段。对PCR扩增片段的酶切样品的预处理和CE条件进行了优化,获得了8种分枝杆菌DNA的限制性内切酶谱图。 DNA片段相对迁移时间的相对标准偏差（RSD）≤3.6%。结果表明,CE的分离效能明显高于AGE,是研究DNA限制性内切酶谱的更有效的检测手段。     

Reduced viscosity polymer matrices for microchip electrophoresis of double-stranded DNA

On a polymethylmethacrylate (PMMA) microchip, double-stranded DNA fragments with a wide size range from 50 bp to 20 kbp were separated by two polymer solutions. One was a hydroxypropylmethylcellulose-4000 (HPMC-4000) solution of 1.3% (w/v) to separate fragments below 590 bp, and another was a mixed four molecular weight poly(ethylene oxide) solution at a total concentration of 0.1% to separate fragments above 520 bp. The widths at half height (wh) of the fragments had a good relationship with their migration times (tR) in both polymer solutions. Such a relationship was suitable for obtaining the wh values of unresolved peaks, calculating the resolution of two adjacent fragments, and optimizing microchip separation matrices. Based on the relativity, a low viscosity medium containing 2% HPMC-50 and 8% glucose was optimized for high-performance separation of a phiX174 HaeIII restriction fragment digest.     

Multiplex polymerase chain reaction analysis of Glu-1 high-molecular-mass glutenin genes from wheat by capillary electrophoresis with laser-induced fluorescence detection

The unique bread-making properties of wheat are closely correlated with composition and quantity of high-molecular-mass (HMW) glutenin subunits encoded by the Glu-1 genes. We report the development of a multiplex polymerase chain reaction (PCR) method to identify bread wheat genotypes carrying HMW glutenin allele composition of Glu-1 complex loci (Glu-A1, Glu-B1 and Glu-D1) by capillary electrophoresis(CE) with laser-induced fluorescence (LIF) detection. Two triplex primer sets of HMW glutenin subunit genes were examined. An automated and rapid CE-LIF technique is helpful in the multiplex PCR optimization process. Two fluorescent intercalating dyes (EnhanCE, and YO-PRO-1) are compared for detection of DNA fragments. Amplified DNA fragments of HMW glutenin Glu-1 genes were well separated both by agarose slab-gel electrophoresis and CE, and revealed minor differences between the sequences of 1Ax2*, 1Axnull, 1Bx6, 1Bx7, 1Bx17 and 1Dx5 genes. Moreover, CE technique requires samples of smaller volumes in comparison to slab-gel electrophoresis, and data can be obtained in less than 20 min. There was a very high concordance in the assessment of the molecular size of PCR-generated DNA markers. Fast and accurate identification of molecular markers of Glu-1 genes by CE-LIF can be an efficient alternative to standard procedure separation for early selection of useful wheat genotypes with good bread-making quality.     

Single strand conformation polymorphism analysis of ras oncogene by capillary electrophoresis with laser-induced fluorescence detector

Single strand conformation polymorphism (SSCP) analysis of the N-ras oncogene was achieved by capillary electrophoresis with a laser-induced fluorescence detector (CE-LIF) using methylcellulose as a molecular sieving agent. The PCR-amplified N-ras oncogene, which is known to have a point mutation at codon 61 in the neuroblastoma, was investigated by CE-LIF combined with SSCP (SSCP-CE-LIF). A mixture of wild- and mutant-type single strand DNA fragments (103 bp) of the N-ras oncogene was separated by buffer solution containing 1.0% methylcellulose and 0.2 microM fluorescent dye (YO-PRO-1) at 25 degrees C. The SSCP-CE-LIF technique gave good resolution for wild- and mutant-type single strand DNA fragments with separation completed within 7 min. SSCP analysis using a CE system with a LIF detector was successfully applied to the detection of the one point mutation on the N-ras oncogene.     

Analysis of double-stranded DNA by capillary electrophoresis using poly(ethylene oxide) in the presence of hexadecyltrimethylammonium bromide

The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by capillary electrophoresis in conjunction with laser-induced fluorescence (CE-LIF) detection using poly(ethylene oxide) (PEO) solution is described. The use of CTAB for improved separation reproducibility and efficiency of DNA has not been demonstrated although it is widely used for controlling the magnitude and direction of electroosmotic flow in CE. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. For the separation of DNA fragments with the sizes ranging from several base pairs (bp) to 2,176 bp, a polymer solution consisting of 0.75% poly(ethylene oxide), 100 mM TB buffer (pH 8.0), 25 microg/mL EtBr, and 0.36 microg/mL CTAB is proper. Using the PEO solution, we separated a mixture of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BglI digest and pBR 328/HinfI digest) within 8 min at -375 V/cm, with the limit of detection of 2.0 ng/mL based on the peak height for the 18-bp DNA fragment. The method is highly efficient (>10(6)plate/m), repeatable (RSD of the migration times <1.5%), and sensitive. In addition, it is convenient to fill a capillary (75 microm in diameter) with such a low-viscosity PEO solution by syringe pushing.     

Capillary electrophoresis with dual laser detection in separation of amplified fragment length polymorphism fragments

We are presenting the application of CE technique with dual‐channel LIF detection for the simultaneous separation of DNA fragments labeled with two different fluorescence dyes. The optimal conditions of the analysis were determined for the separation of amplified fragment length polymorphism (AFLP) fragments labeled with 5′‐6‐carboxyfluorescein (6‐FAM) and the DNA size standard labeled with sulfoindocyanine succinimidyl ester (Cy‐5). CE equipped with both argon ion and diode lasers is a good alternative for sequencers and might be applied in analyses of PCR products generated by various fingerprinting methods.     

Screening of mixed poly(ethylene oxide) solutions for microchip separation of double-stranded DNA using an orthogonal design approach

We describe a quick and systematic optimization of molecular weights (MWs) and concentrations of a series of mixed poly(ethylene oxide) (PEO) matrices for separating specific double-stranded DNA fragments on polymethylmethacrylate-based microchips by using an orthogonal design (ORD) approach. The mixed matrices are composed of PEOs in four MW ranges (M(w) 8 x 10(6), 1 x 10(6), 4 x 10(5), and 1 x 10(5)) with varying concentration ratios. In the mixed solutions, PEO with an intermediate MW of 4 x 10(5) is found to be a dominant factor for separating small DNA fragment pairs (e.g., 82 and 88 bp), while PEO with a high MW of 8 x 10(6) plays an important role in separating intermediate and large fragments (e.g., 271 and 281 bp, 506 and 517 bp, 7 and 10 kbp). High-concentration PEO mixtures give better resolution for short fragments, while dilute PEO mixtures show better resolution for long fragments. The optimized matrices are suitable for high-resolution separation of multiplex polymerase chain reaction-amplified products and restriction digest fragments ranging in size from 20 bp to 40 kbp within 4 min at a constant field strength of 177 V/cm. The experimental results indicate the robustness and speediness of the ORD to screen the contribution of PEO MWs and to tune optimally the PEO concentration ratio of different MWs with reference to the performance of specific DNA fragments separated.     

Chemical synthesis of a structural gene coding for trichosanthin

A structural gene (750 bp), which codes for a type I ribosome inactivating protein, trichosanthin, has been designed according to the codon usage of highly expressed gene in E. coli and chemically synthesized. In the synthesized gene, twenty-seven unique restriction sites were evenly dispersed with an average distance between two adjacent sites less than 50 bp to facilitate a systematic investigation on structure-functional relationship of this protein by site-directed muta-genesis. To synthesize it, the whole gene was divided into three large fragments (EP, PN and NH) which were assembled from several chemical synthetic oligonucleotides by enzymatic method. The assembly of both the fragment EP from six oligonucleotides (A-F) and the fragment PN from four oligomers (G-J) was catalyzed by T4 DNA ligase in using the single stranded DNA method [Chen, H.-B. et al, Nucl. Acids Res., 18, 871(1990)]. And fragment NH was formed from three duplexes K, L and M by the classical double stranded DNA method. Finally,     

Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.     

Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis

Three methods [capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)] were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157. The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively. Subsequent CE analysis was carried out. Separation time was 4 min. SSCP, which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA. Two genes could be analyzed in approximately 18 min. CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA. The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2. The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP. Separation time was 6 min. These techniques are suitable for the detection and the identification of O157.