用未涂敷毛细管电泳分离DNA片段

用内壁未涂敷毛细管，以羟乙基纤维素的无胶筛分介质，在６ｍｉｎ内分离了λＤＮＡ／ＥｃｏＲＩ＋ＨｉｎｄⅢ片段，探讨了ＤＮＡ片段在未涂敷毛细管中的分离机理，讨论了羟乙基纤维素和缓冲溶液浓度及电压对分离的影响，考察了ＤＮＡ片段迁移时间的重现性。     

DNA片段分选微流控芯片发展现状

新一代测序速度越来越快,费用不断降低,但整个流程中还有不少瓶颈.最耗时且影响测序准确度和重复性的环节之一,就是在文库制备过程中,对打断的大量基因组DNA片段进行基于传统凝胶电泳的手工操作筛选.近几年市场上出现了几种目标片段自动分选仪器,并迅速被多家国际知名测序中心引入测试,从而引起了广泛关注.本文评述了DNA片段分选发展经历,包括凝胶电泳、毛细管电泳、特别是微流控芯片在分选精度和通量上的不断提升,并简要论述了DNA分选目前尚存在的问题.     

Analysis of Sources of Error in Quantitation of Purified DNA Fragments and Unpurified PCR Products by DNA Microchip Electrophoresis

We have investigated the accuracy and reproducibility of DNA quantitation on the DNA Lab-Chip and the relationship of these to the size and concentration of the DNA fragments. We found that quantitation of small DNA fragments, i.e. less than 200 bp, suffers from high relative error which can be improved by using an internal standard of similar size to the sample. The effects of trace chloride ion on quantitation error and sensitivity of the DNA Lab-Chip were also studied, and it was revealed that 0.2 mM chloride ion reduces quantitation sensitivity by 30% and increases the relative error. We also studied the effects of purification on quantitation errors in analysis of PCR products from cloning vector pUC118 and showed that use of an unpurified sample reduces chip sensitivity by 25%.Dedicated to Professor K. Jinno on the occasion of his 60^th birthday.Revised: 9 December 2004 and 17 January 2005     

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）。     

无胶筛分毛细管电泳分离盐生盐杆菌DNA片段

 由羟乙基纤维素和聚吡咯烷酮混合组成筛分介质 ,在涂敷聚硅氧烷的毛细管柱上 ,研究了LambdaDNA/EcoRⅠ +HindⅢ片段分离的最佳条件。实验表明 ,混合筛分介质与单一的羟乙基纤维素筛分介质相比 ,改变了筛分介质的孔径大小 ,抑制了毛细管壁对DNA的吸附 ,从而改善了分离 ,并首次在同一条件下将所含的 13个片段完全分离。方法简便、快速 ,曾应用于两组盐生盐杆菌DNA片段的分离及其碱基对数目的推测。     

毛细管电泳聚乙烯吡咯烷酮与羟乙基纤维素混配无胶筛分介质分离DNA片段

本文报道了毛细管电泳聚乙烯吡咯烷酮与羟乙基纤维素混配无胶筛分介质分离较短的 p GEM7Zf(+) Hae DNA片段 (DNA长度为 1 8～ 675bp)。研究表明 ,在 1 %的羟乙基纤维素无胶筛分介质中 ,加入 2 %的聚乙烯吡咯烷酮能显著提高 DNA片段的分辨率和分离效率。在混配无胶筛分介质中 ,聚乙烯吡咯烷酮有两种作用 ,一是动态涂渍 ,降低毛细管内壁对 DNA片段与 DNA荧光插入试剂的吸附 ,改善分离效率 ;二是两种不同长度、性质的线性高分子能形成更为致密的“缠绕网络”,有利于较短的 DNA片段电泳分离。     

Further Study on Separation of DNA Fragments by Capillary Electrophoresis by Quasi-interpenetrating Network of Polyacryamide and Polyvinylpyrrolidone with UV Detection

Quasi-interpenetrating network of polyacrylamide （PAA） and polyvinylpyrrolidone （PVP） had been successfully used for single-base resolution of double-stranded DNA （0.76 for 123 bp/124 bp） and single-stranded DNA fragments （0.97 for 123 b/124 b） with UV detection. This quasi-IPN （interpenetrating network） sieving matrix showed low viscosity （23.5 mPa·s at 25 ℃） and decreased with increasing temperature. This polymer also exhibited dynamically coating capacity and could be used in the uncoated capillary. The effects of temperature and electric field strength on the DNA separation of quasi-IPN matrix were also investigated and found that the temperature and electric field strength could markedly affected the mobility behavior of DNA fragments. This polymer matrix has also applied to separate the bigger DNA fragments by capillary electrophoresis with UV detection. Under the denaturing conditions, this matrix separated the samples with last fragment of 1353 base in 40 rain, in which the doublet of 309/310 base was partial separated and the resolution was 0.88.     

Investigation of a Homologous Series of Fatty Acids by TLC. Part III. Application of Terms Describing the Separation of Homologous Series of Saturated Fatty Acids in TLC

JPC – Journal of Planar Chromatography – Modern TLC - Fatty acids from pentanoic to tricosanoic have been separated on RP-18 HPTLC plates, with and without concentrating zone, with...     

高效液相色谱非多孔型阴离子交换柱分析DNA片段

建立了高效液相色谱非多孔型阴离子交换柱分离ＤＮＡ片段的方法,用ＴＳＫｇｅｌＤＥＡＥ－ＮＰＲ柱、Ｔｒｉｓ－ＨＣｌ缓冲液（ｐＨ９．０）、１．０ｍｏｌ／ＬＮａＣｌ多阶式线性梯度洗脱,分析了核酸分子量参照物ｐＢＲ３２２ＤＮＡ－ＨａｅⅢ,ＬａｍｂｄａＤＮＡ－ＨｉｎｄⅢ及乙肝病毒基因ＰＣＲ产物,探讨了梯度、流速对分离的影响,方法简便、快速、分辨率高     

聚环氧乙烷无胶筛分毛细管电泳分离宽分子量范围DNA片段

在无胶筛分毛细管电泳中,以聚环氧乙烷为筛分介质,用硅烷化处理的毛细管柱（31.2 cm×75 μm有效长度21.0 cm）分离DL5000 DNA Marker（DNA长度为100~5000 bp）,研究筛分介质浓度、缓冲液pH、分离电压和溴化乙锭浓度对分离双链DNA片段的影响,优化出分离100~5000 bp DNA片段的最佳条件。毛细管电泳的最佳条件为PEO浓度0.5%、缓冲液pH值8.0、电压12 kV、溴化乙锭浓度3.0 μg/mL。此条件下,对山梨醇脱氢酶基因（SDH）和乙烯受体基因（ETR1）的聚合酶链式反应（PCR）扩增产物同时检测,分离、鉴定效果良好。     

高效液相色谱-四极杆飞行时间质谱分析紫甘蓝和羽衣甘蓝中花色苷

应用高效液相色谱-电喷雾/四极杆飞行时间串联质谱联用技术分析了紫甘蓝和羽衣甘蓝中的花色苷成分.选用Agilent TC-C18色谱柱(250 mm×4.6 mm×5 μm),二元线性梯度洗脱,柱后流出液采用电喷雾四极杆飞行时间质谱的正、负离子模式进行检测.根据一级质谱的分子离子和二级质谱碎片离子,获得化合物的准确分子量...     

Neuroprotective effects of Tilia americana var. mexicana on damage induced by cerebral ischaemia in mice

Tilia americana var. mexicana (T. americana) is a plant widely used in Mexico for its medicinal properties on the central nervous system. In the present study, we designed a protocol to investigate the neuroprotective effects of non-polar and polar extracts of T. americana on damage induced by cerebral ischaemia in mice. Vehicle or extracts were administered immediately after ischaemia. Functional neurological deficit, survival percentage and infarct area were determined in each experimental group. Results showed that groups treated with non-polar or polar extracts of T. americana had increased survival rate, improved neurological deficits and diminished the infarct area in relation to the ischaemic group. In conclusion, this study confirms the neuroprotective activity of T. americana, suggests a possible synergism between non-polar and polar constituents and supports its potential as a useful aid in the clinical management of stroke.     

Investigation of Homologous Series of Fatty Acids by TLC. Part IV. Separation on RP18 Plates with Ternary Mobile Phases

JPC – Journal of Planar Chromatography – Modern TLC - Fatty acids from heptanoic to eicosanoic have been separated on RP-18 HPTLC plates, with and without concentrating zone, with...     

Synthesis of a teichoic acid fragment of bacillus var.niger w.m. by a phosphotriester approach

Three properly-protected derivatives, one non-terminal and two terminal units, of 3-0-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-sn-glycerol have been joined together by two interglyceridic (2→1) phosphotriester linkages to afford, after removal of all protective groups, a teichoic acid fragment.     

Capto™ Resins for Chromatography of DNA: A Minor Difference in Ligand Composition Greatly Influences the Separation of Guanidyl-Containing Fragments

The differences in chromatographic behaviour of individual deoxynucleotides as well as small single-stranded and double-stranded DNA molecules have been examined for two resins from the Capto family: Capto Adhere and Capto Q ImpRes. Capto Adhere carries a multimodal ligand which combines strong anion with aromatic recognition, while Capto Q ImpRes is a strong anion exchanger with a chemically similar ligand, but without a phenyl group. The intrinsic differences between single- and double-stranded DNAs are related to charge, hydrophobicity, size and three-dimensional structure. These variations in biophysical properties have been utilized for comparative separations on these two resins. All deoxynucleotides and DNAs tested bound strongly to the chromatographic materials and could be eluted by a linear gradient of increasing NaCl concentration. Capto Q ImpRes provided a recognition for guanylate bases when samples of deoxynucleotides or poly(dG) were examined. This recognition was not observed for Capto Adhere. Another pronounced difference between the resins was observed in the inverted elution of ss- and dsDNA, where ssDNA eluted at 2.88 M NaCl on Capto Adhere, while on Capto Q ImpRes ssDNA eluted already at 1.47 M NaCl. This behaviour can be linked to the presence of the more hydrophobic phenyl group in Capto Adhere, leading to stronger retention of ssDNA molecules, which have a more hydrophobic character due to a higher degree of base exposure.

     

Determination of 6-methyladenine in DNA by high-performance liquid chromatography.

A method for the determination of 6-methyladenine (6MA) by high-performance liquid chromatography (HPLC) has been developed. DNA bases were separated by using the strong cation-exchange resin Zipax SCX. Purine bases were obtained by hydrolysis and dialysis of DNA and analysed by HPLC. 6MA in DNA from Escherichia coli was determined by the proposed method. It is suggested that the method could be applicable to analyses of 6MA from other biological sources.     

Biosynthesis of the validamycins: identification of intermediates in the biosynthesis of validamycin A by Streptomyces hygroscopicus var. limoneus.

To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in (2)H-, (3)H-, or (13)C-labeled form and fed to cultures of Streptomyces hygroscopicus var. limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintillation counting, (2)H- or (13)C NMR or selective ion monitoring mass spectrometry (SIM-MS) techniques. The results demonstrate that 2-epi-5-epi-valiolone (9) is specifically incorporated into 1 and labels both cyclitol moieties. This suggests that 9 is the initial cyclization product generated from an open-chain C(7) precursor, D-sedoheptulose 7-phosphate (5), by a DHQ synthase-like cyclization mechanism. A more proximate precursor of 1 is valienone (11), which is also incorporated into both cyclitol moieties. The conversion of 9 into 11 involves first epimerization to 5-epi-valiolone (10), which is efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of 2-epi-valienone (15) is also observed. Reduction of 11 affords validone (12), which is also incorporated specifically into 1, but labels only the reduced cyclitol moiety. The mode of introduction of the nitrogen atom linking the two pseudosaccharide moieties is not clear yet. 7-Tritiated valiolamine (8), valienamine (2), and validamine (3) were all not incorporated into 1, although each of these amines has been isolated from the fermentation, with 3 being most prevalent. Demonstration of in vivo formation of [7-(3)H]validamine ([7-(3)H]-3) from [7-(3)H]-12 suggests that 3 may be a pathway intermediate and that the nonincorporation of [7-(3)H]-3 into 1 is due to a lack of cellular uptake. We thus propose that 3, formed by amination of 12, and 11 condense to form a Schiff base, which is reduced to the pseudodisaccharide unit, validoxylamine A (13). Transfer of a D-glucose unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge between the two pseudosaccharide units are also discussed.     

Changes in the secondary structure of HMGB1 protein bonded to DNA

The stage of noncooperative interaction of the chromosomal nonhistone protein HMGB1 with DNA has been studied by spectroscopic methods and gel retardation. It was found that complexation was accompanied by compaction of the DNA molecule over a wide range of protein/DNA ratios in the complex. A circular dichroism study showed that the binding with DNA changed the secondary structure of the HMGB1 protein. Changes in the structure of the protein start under the conditions of an excess of binding sites on DNA and end at a ratio of ∼40–50 base pairs per protein molecule, the α-helicity of HMGB1 in the complex increasing by 20% compared with the free state. It is believed that the change in the secondary structure of HMGB1 during the binding with DNA underlies the mechanisms of the various functions of this protein in the cell.     

玻璃基质微流控芯片用于DNA片段的分离和C677T基因突变的快速检测

基于微加工方法,在一般实验条件下,成功地制备了玻璃芯片。利用水溶性聚合物对玻璃通道进行动态修饰,从而抑制了DNA分子的吸附作用,并成功地用于DNA片段的分离和四氢叶酸还原酶基因(MTHFR)中C677T的突变检测。