毛细管电泳—激光诱导荧光法分离检测DNA片段与基因扩增产物

以羟丙甲基纤维素和非交联聚丙烯酰胺溶液为筛分介质，将毛细管电泳－激光诱导荧光法用于ＤＮＡ片段及基因扩增产物的分离检测。探讨了非胶筛分介质中高分子化合物的浓度、电解质的浓度、内插试剂用量等对ＤＮＡ片段分析检测的影响；考察了ＤＮＡ片段迁移时间和峰面积的重现性及ＤＮＡ片段定量检测的关系，建立了一种快速、灵敏的ＤＮＡ片段及基因扩增物分离检测方法。     

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。     

荧光偏振技术分析全血XPD基因单核苷酸多态性的研究

将荧光偏振与非对称基因扩增技术联用,建立了可用于检测全血XPD基因单核苷酸多态性的新方法。用不等量(1∶5)的XPD基因上、下游引物对含单核苷酸多态性位点的目的片段进行非对称扩增,再用两种单核苷酸多态性序列特异的荧光标记探针对扩增产物进行检测。由于扩增得到的单链片段能够与各自不同的荧光标记探针特异结合,使荧光标记分子的分子量增加,偏振值(FP)增高。通过检测增高的FP值,可确定目的片段单核苷酸多态性。采用本方法对98例全血的XPD基因第751位密码子进行了单核苷酸多态性分析,并与传统的荧光偏振检测方法进行了比较,取得满意结果。     

DNA芯片技术与脱氧核糖核酸序列分析

人类基因组计划的实施,有力地促进了ＤＮＡ序列分析技术的发展。ＤＮＡ芯片综合运用了固相合成化学、照相平版印制技术以及激光共聚焦扫描等技术,能够同时扫描分析多基因乃至基因组,可广泛应用于基因的多态性分析、基因定位、表达水平的监测以及遗传病的诊断等领域,是对传统的ＤＮＡ分析技术的一次重大突破。本文介绍了ＤＮＡ芯片技术的基本原理并对其应用作一简要综述。     

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DN …

本文报道了毛细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ｐＧＥＭ７Ｚｆ（＋）－ＨａｅⅢＤＢＡ片段（ＤＮＡ长度为１８～６７５ｂｐ）。研究表明,在１％的羟乙基纤维素无胶筛分介质中,加入２％的聚乙烯吡咯迷酮能显著提高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     

变性无胶毛细管筛分电泳分析聚合酶链式反应产物

在分离Ｙ染色体性别决定区,人１１号染色体短β－珠蛋白基因部分序列扩增产物和ＰＢＲ３２２／ＨａｅⅢ标准片段的混合样品时,ＳＲＹ和１０８／Ｂ的迁移时间均产生较大的偏差,在尿素变性存在的无胶筛分体系中迁移时,这种迁移时间的偏差得以消除,这意味着尿素性剂的存在消除了ＤＮＡ空间结构对迁移的影响,ＤＮＡ在此筛分介质中的迁移只与其片段长度有关。     

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     

用盐酸阿霉素作为嵌入剂石墨电极伏安法识别测定DNA片段的研究

用盐酸阿霉素作为嵌入剂石墨电极伏安法识别测定ＤＮＡ片段的研究方禹之,刘盛辉,何品刚（华东师范大学化学系,上海,２０００６２）关键词ＤＮＡ,嵌入剂,盐酸阿霉素,伏安法核酸分子杂交技术是近年来发展起来的检测ＤＮＡ分子的一种重要手段,目前主要采用的放射性同...     

在丝状噬菌体主要外壳蛋白上展示外源多肽的载体

利用基因工程手段,构建了两个用于在丝状噬蓖体主要外壳蛋白表面展示外源多肽的载体,这类载体可以使编码外源多肽的ＤＮＡ片段定向克隆到外壳蛋白的Ｎ末端ＤＮＡ序列中或附近,被修饰的外壳蛋白将分布于野生型丝状噬菌体外壳蛋白单位中间。     

稀土化合物对核酸分子完整性的影响

由于稀土元素具有独特的理化性质 ,被广泛应用于工业、农业、医药卫生等领域。1993年日本东京大学研究人员报道了Ｃｅ ,Ｔｍ ,Ｌａ的有机络合催化剂可使核酸水解 ,从而达到对病毒和癌细胞ＲＮＡ和ＤＮＡ的特定切割 ,认为如果稀土元素应用于防治癌症和艾滋病取得成功 ,将具有非常重大的意义[1～ 4 ] 。目前 ,国内外所做研究均为 4 0个核苷酸左右或更短片段的线性人工合成的ＤＮＡ或ＲＮＡ[5～ 7] ,对序列更长的天然ＤＮＡ及ＲＮＡ的研究还有待于进一步深入。本文研究了不同剂量的 3种稀土化合物对微生物、动物及人的ＤＮＡ和ＲＮＡ分子完整性…     

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.     

毛细管电泳——基因突变及多态性分析新方法

着重介绍基因突变及多态性分析方法以及毛细管在电泳在该领域中的应用。主要包括单链构象多态性分析,变性梯度及温度梯度电泳,杂合子分析,限制性片段多态性分析,等位基因特异性扩增,核酸杂交,引物扩展及小卫星和微卫星分析。     

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.     

Use of capillary electrophoresis for high throughput screening in biomedical applications. A minireview

Diagnosis of inherited diseases or cancer predispositions frequently involves determination of specific mutations or polymorphisms. The number of characterized monogenetic and polygenetic diseases is significantly rising every year. As a result, an increasing number of patient samples with a rising complexity of genetic diseases require molecular diagnostics. In order to apply genetic analyses to large groups of patients or population screening, automation of a sensitive and precise method is highly desirable. Capillary electrophoresis (CE) facilitates the development of methods which can rapidly process large number of patient samples in an automated fashion. In contrast, conventional techniques including Southern blotting, sequencing or standard gel electrophoresis are time consuming, cost ineffective and require substantial amounts of each specimen. Robustness, ease of operation, good reproducibility and low cost are the main advantages of CE. Currently, most protocols adapted to automated CE represent (i) analyses of DNA fragment length or DNA restriction patterns (RFLP), (ii) analyses of single-strand conformation polymorphism (SSCP) and (iii) microsatellite analyses. Recently, automated detection of variations in the FRAXA (CGG)n region (fragile X syndrome), LDL receptor gene, p53 gene, MTHFR (methylenetetrahydrofolate reductase) gene, HFE gene and others has been established on CE systems. These applications clearly demonstrate the suitability of CE for high throughput screening in medical applications.     

High-throughput screening of genetic mutations/polymorphisms by capillary electrophoresis

Genetic mutations/polymorphisms analyses play a great role in genetic and medical research, and clinical diagnosis. Most conventional methods for genetic assay are based on slab gel electrophoresis that is both labor-intensive and time-consuming. Recently, capillary electrophoresis (CE) has been used for genetic analysis instead of conventional slab gel electrophoresis. This technique can be automated and is characterized by short analysis time, small sample and reagents requirements, and high separation efficiency. CE has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes, and has shown a great potential for genetic mutation/polymorphism screening of large numbers of clinical samples. In this article, an overview of the fundamental aspects of mutation/polymorphism assay methods in combination with CE is given and some key applications are summarized.     

Capillary electrophoresis ribosomal RNA single-stranded conformation polymorphism: a new approach for characterization of low-diversity microbial communities

Capillary electrophoresis (CE) has been the principle system for nucleic acid analysis since the early 1990s due to its inherent advantages such as fast analysis time, high resolution and efficiency, minimal sample requirement, high detection sensitivity, and automation. In the past few decades, microbial community fingerprinting methods such as terminal restriction fragment length polymorphism and single-stranded conformation polymorphism (SSCP) have migrated to CE to utilize its advantages over conventional slab gel electrophoresis. Recently, a gel-based direct rRNA fingerprint method was demonstrated. Different from other existing microbial community characterization approaches, this novel approach is polymerase chain reaction free and capable of providing information on the relative abundance of rRNA from individual phylotypes in low-diversity samples. As a gel-based method, it has a long analysis time and relatively large reagent and sample requirements. Here, we addressed these limitations by transferring the RNA fingerprint approach to the CE platform. Analysis time significantly improved from 24?h to 60?min, and the use of a fluorescently labeled hybridization probe as the detection strategy decreased the sample requirement by ten-fold. The combination of fast analysis time, low sample requirement, and sensitive fluorescence detection makes CE-RNA-SSCP an appealing new approach for characterizing low-diversity microbial communities.     

Identification of individual DNA molecule of Mycobacterium tuberculosis by nested PCR-RFLP and capillary electrophoresis

The improvement of sensitivity and differentiation in rapidly identifying a small amount of mycobacteria in sputum has significant implications for reducing tuberculosis transmission. We previously applied the conventional PCR and capillary electrophoresis (CE) to establish the restriction fragment length polymorphism (RFLP) pattern of mycobacterial 65-kDa heat shock protein (hsp65) gene from colony specimens. However, the previous analysis did not provide enough sensitivity for sputum specimens in which the limitation of analysis might be hindered by PCR inhibitors and primer-dimers formation during amplification. In the current study, nested PCR (nPCR) had been redesigned for PCR-RFLP analysis (PRA) of mycobacterial hsp65 gene using CE. The results show both Mycobacterium tuberculosis complex and mycobacteria other than tuberculosis could be identified in the presence of PCR inhibitors. The interference due to primer-dimers was also minimized. Based on the Poisson distribution, the repeatability of single DNA molecule detection was greatly affected by sampling probability and might be improved significantly by increasing the sample loading. The PRA using nPCR and CE is not only able to detect the individual mycobacterial DNA molecule but also potentially differentiate the species.     

Structural factors determining DNA length limitations in conformation-sensitive mutation detection methods

Numerous mutations and polymorphisms in human genes remain to be identified using reliable methods. Of the available mutation scanning methods those dependent on structural change-induced mobility shifts are highly effective. Their efficiency is, however, DNA length-sensitive and the reasons for that are poorly understood. In this study, we explain why scanning genes for mutations is less effective in longer DNA fragments, and reveal the factors which are behind this effect. We have performed a systematic analysis of the same sequence variants of exon 11 of the BRCA1 gene in DNA fragments of three different lengths using the combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (DA) by capillary electrophoresis (CE). There are two major structural factors responsible for the reduced mutation detection rate in long amplicons. The first is increased contribution from other secondary structure modules and domains in longer fragments, which mask the structural change induced by the mutation. The second is higher frequency of single-nucleotide polymorphisms (SNPs) including common polymorphisms in longer fragments. This makes it necessary to distinguish the structural effect of the mutation from that of each polymorphic variant, which is often difficult to achieve. Taking these factors into account, an efficient scanning of genes for sequence variants by conformation-sensitive methods may be performed.     

A simple and precise diagnostic method for spinal muscular atrophy using a quantitative SNP analysis system

A simple and precise diagnostic method for spinal muscular atrophy (SMA) using high‐resolution CE‐based single‐strand conformation polymorphism (CE‐SSCP) was developed in this study. SMA is a common genetic disorder caused by an abnormality in the relative copy numbers of SMN1 and its centromeric copy SMN2, which differ only in two nucleotides, namely at exons 7 and 8. Therefore, the precise discrimination of the differences in sequence as well as their relative quantities is crucial for the diagnosis of SMA. Multiplex ligation‐dependent probe amplification and sequence‐sensitive DNA separation using hydroxyethyl cellulose and hydroxypropyl cellulose blended polymer matrix are currently the available methods used in the diagnosis of SMA. However, these methods are limited by their extended hybridization step and low resolution. In this study, the simultaneous discrimination of SMN exons 7 and 8 was successfully demonstrated using high‐resolution CE‐SSCP. Unlike the previously reported alternative method, single base differing amplicons were baseline‐separated because of its extraordinary resolution, thus providing accurate and precise quantification of each paralog.     

Single nucleotide polymorphism detection in the hMSH2 gene using conformation-sensitive CE

CE allows for highly reproducible analysis of DNA fragments which can be used to detect DNA mutations including SNPs. We have utilized a simple and direct CE analysis method for SNP analysis called conformation-sensitive CE (CSCE), based on the principle of single nucleotide different to produce conformational changes in the mildly denaturing solvent system. This method was applied to analysis of a mutation in the promoter region of the hMSH2 gene. This gene belongs to the human DNA mismatch repair system, which is responsible for recognizing and repairing mispaired nucleotides, and mutations in the hMSH2 gene are known to cause hereditary nonpolyposis colorectal cancer (HNPCC). PCR fragments generated from the promoter region of the hMSH2 gene, displaying either a C/C homozygote, C/T heterozygote, or T/T homozygote genotype, did not require further pretreatment before electrokinetic injection. The CE separation, using a 1xTris-borate-EDTA (TBE) buffer containing 3% w/v hydroxylethyl cellulose (HEC) and 6 M urea, was performed under reverse polarity with a separation temperature of 15 degrees C. The genotypes of 204 healthy volunteers and 13 colorectal cancer patients were determined using CSCE, and the results confirmed by DNA sequencing. While the CSCE separations were shown to be highly reproducible and sensitive for screening large populations, no correlation was observed between cancer patients and this hMSH2 gene polymorphism.