单链构象多态性毛细管电泳分析研究进展

基因突变的检测在临床疾病诊断中起十分重要的作用.单链构象多态性(Single Strand Conform ationPolym orphism,SSCP)分析是检测突变最流行的方法之一.SSCP分析与毛细管电泳(Capillary Electrophoresis,CE)相结合的技术更是具有灵敏度高、花费低、简单、快速的优点.目前,这项技术已应用于人类原癌基因、抑癌基因以及其它致病基因的突变检测.主要综述了各种参数对CES-SCP分析的影响以及CES-SCP分析技术将来的发展方向.     

HCV准种单链构象多态现象的检测

单链构象的多态现象(SSCP)是根据PCR技术广泛用于检测不同DNA序列的一门技术，由遗传学家开发的致突变检测，最近已采用于病毒基因组准种的分析，如丙型肝炎病毒(HCV)。由于该技术严格的标准化及其检测的局限性使之很难推广，HCV高变异区准种内的变异体被克隆到Puc　119，引起质粒定量，这种变种可用作SSCP最佳模板，所研究的变量有许多检测到的变异体，少数变异体检测的敏感性，电泳温度、单链DNA产生的方法、PCR循环次数的影响以及与校读能力有关的DNA多聚酶的应用。结果表明，它是能在准种内至少测出5种变异体的最佳方法，并能检出低至2％水平的准种内的变异体，电泳在室温18小时其重复性最好，使用Taq多聚酶单个引物作20次循环产生的单链DNA给准种构成一个精确的映象，Pfu多聚酶的应用减低了检测较小的带型的敏感性。SSCP法提供了评价HCV准种一个精确的工具。     

单链构象多态性分析与应用

单链构象多态性(single-strand conformational poly-morphism,SSCP)是一种简单、方便,且十分有效的分析核酸(DNA或RNA)变异的方法和技术,是Orita等在1989年首先建立的,其基本原理是单链核酸于非变性聚丙烯酰胺凝胶电泳的迁移率与核酸的一级结构和构象密切相关,核酸序列的变异,甚至单个碱基的改变,都可能改变核酸片段的构象,从而改变核酸的迁移速率,因此可以将“变异DNA”与正常的DNA区分开来。尽管目前还难以根据SSCP电泳迁移率的     

单链构象多态性（SSCP）及其在昆虫学中的应用

单链构象多态性（SSCP）能够快速、灵敏地检测基因的点突变,其应用范围日益扩大。本文重点综述了其在昆虫学研究中的作用。 bstract:Single-strand conformation polymorphism (SSCP) can detect point mutation quickly and sensitively and its scope of application is enlarged everyday.In this paper,its important roles in entomology are summarized.     

甘油对单链构象多态性分析的影响

单链构象多态性(single strand conformation poly-morphism,SSCP)分析是以PCR技术为基础检测DNA多态性的一种方法。其基本原理是:单链DNA在进行不含变性剂的中性聚丙烯酰胺凝胶电泳(PAGE)时,迁移率除与DNA单链的长短有关外,更主要取决于DNA单链所形成的具有一定空间结构的构象。这种构象由DNA单链碱基顺序决定,其稳定性靠分子内部局部顺序的相互作用来维持。相同长度的单链因其顺序的不同,甚至单个碱基的不同,所形成的构象不同,导致迁移率的变化而出现泳动变位(mobili-ty shift)。该方法自建立起来不断地发展、完善,应用范围也日益广泛。特别是在人类癌基因和抑癌基因突变的检测、基因诊断、连锁分析、基因作图等领域成效突出。     

非同位素PCR－单链构象多态性技术的建立和应用

PCR-单链构象多态性技术(SSCP)问世以来,成为研究基因突变的工具.特别是在分子肿瘤学研究中,广泛应用于癌基因、抑癌基因突变的研究.常规PCR-SSCP采用同位素标记PCR产物,测序板电泳分离突变,在操作和费用上有种种局限,文章建立了一种非同位素PCR-SSCP技术:通过不对称PCR获得单链,普通PAGE分离,经银染检出突变.用这种方法,还研究了四株鼻咽癌细胞株CNE1,CNE2,HK1和SUNE1中肿瘤抑制基因p53基因突变.证实CNE1,CNE2在exon8,HK1在exon5有突变,并发现新建立的细胞株SUNE1在exon8有突变.     

毛细管电泳-单链构象多态分析检测胃癌中p16基因突变

目的:建立快速高效检测胃癌患者胃癌组织及癌旁正常组织中p16基因突变的方法。方法:采用PCR扩增p16基因第二外显子易发生突变片段,扩增样品纯化后经95℃变性;以毛细管电泳(CE)分析法结合单链构象多态性(SSCP)对60例胃癌患者p16基因突变情况进行分析。结果:分析结果表明只有3例低分化腺癌患者存在基因突变,测序表明p16基因第二外显子碱基序列AGAC发生碱基A丢失。结论:p16基因突变可能导致胃癌的发生,但不起主导作用;CE-SSCP分析方法具有快速、灵敏、准确的特点,可用于胃癌组织中p16基因的突变分析。     

PCR-SSCP的效果分析

PCR-SSCP是一种以PCR为基础的单链构象多态性分析技术,是DNA已知突变的检测或未知变异分析中常用和实用的技术之一。影响SSCP试验效果的因素有很多,本研究主要对凝胶浓度和是否添加甘油两个因素进行分析与探讨。结果表明,凝胶浓度12%和添加甘油终浓度10%的条件下可以得到满意的结果。     

PCR—单链构象多态性分析对p53基因点突变检测的研究进展

单链构象多态性（ＳＳＣＰ）对分析基因的突变是一种有效的手段,并具有其独特的优点。ＰＣＲ与ＳＳＣＰ结合后检测灵敏度更高,而在许多类型的肿瘤都存在有ｐ５３抑癌基因的突变,章综述了ＰＣＲ－ＳＳＣＰ分析技术检测ｐ５３基因突变的进展。     

肠杆菌4.5SRNA基因单链构象多态性的比较研究

本文对大肠杆菌(Escherichia coli)不同菌株和肠杆菌科(Enterobacteriaceae)不同属其他三种菌株,即普通变形菌(Proteus vulgaris)、粘质沙雷氏菌(Serratia marcescens)和产气肠杆菌(Enterobacter aerogenes),分别进行4.5S RNA基因聚合酶链式反应(PCR),然后对扩增产物作依赖于序列的单链构象多态性(SSCP)分析.实验结果表明,上述细菌4.5S RNA基因的大小和正链构象均无可觉察的差异,仅产气肠杆菌的负链构象有明显不同.由此可见4.5S RNA基因在进化上相当保守,产气肠杆菌4.5S RNA基因的序列虽有改变,仍能维持其有义链的基本构象.     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

DNA Single Strand Breaks by Aromatic Nitroso Compounds in the Presence of Thiols

Aromatic nitroso compounds, nitrosobenzene (NB), N, N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.     

Heterogeneity of HLA-G Genes Identified by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP)

A genomic HLA-G clone named 7.0E was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA-G genomic clones and two cDNA clones previously described. The DNA sequences of α1 and α2 domains of the HLA-G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3′ untranslated region of 7.0E compared with the originally described HLA-G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA-G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.0E-type were more heterogeneous than those of the G6.0- type. The 14 bp deletion is present only in the G6.0-type of HLA-G alleles among HLA class I genes. Thus it was suggested that G6.0 alleles were generated after diversification of the HLA-G.     

Interaction of Microwaves and a Temporally Incoherent Magnetic Field on Single and Double DNA Strand Breaks in Rat Brain Cells

The effect of a temporally incoherent magnetic field noise on microwave-induced DNA single and double strand breaks in rat brain cells was investigated. Four treatment groups of rats were studied: microwave-exposure (continuous-wave 2450-MHz microwaves, power density 1 mW/cm², average whole-body specific absorption rate of 0.6 W/kg), noise-exposure (45 mG), microwave + noise-exposure, and sham-exposure. Animals were exposed to these conditions for 2h. DNA single- and double-strand breaks in brain cells of these animals were assayed 4h later using a microgel electrophoresis assay. Results show that brain cells of microwave-exposed rats had significantly higher levels of DNA single- and double-strand breaks when compared with sham-exposed animals. Exposure to noise alone did not significantly affect the levels (i.e., they were similar to those of the sham-exposed rats). However, simultaneous noise exposure blocked microwave-induced increases in DNA strand breaks. These data indicate that simultaneous exposure to a temporally incoherent magnetic field could block microwave-induced DNA damage in brain cells of the rat.     

三硝基甲苯致晶状体上皮细胞DNA损伤作用的研究

本文通过出生后７天和２４个月两组大鼠晶状体与２０％三硝基甲苯（ＴＮＴ）甘油：水（８：２）混悬液体外培养,培养箱通５％ＣＯ２,恒温３７℃,观察ＴＮＴ对晶状体上皮细胞ＤＮＡ的损伤程度,结果以反映单链ＤＮＡ断裂强度的Ｆ６３０／Ｆ５３０比值表示。ＨＰＬＣ分析发现经体外ＴＮＴ孵育的晶状体内含有ＴＮＴ及其代谢产物;当共同培养９６ｈ后,Ｆ６４０／Ｆ５３０比值随着ＴＮＴ剂量增加而增大,达到４０μＬ（１１．７４μｍｏｌ／Ｌ）时趋向平稳;当用ＴＮＴ浓度为４．４０μｍｏｌ／Ｌ的相同剂量培养时,４８小时后单链ＤＮＡ断裂最为严重Ｆ６３０／Ｆ５３０比值为１．５０,明显高子正常对照组（Ｐ＜０．０１）;幼鼠单链ＤＮＡ断裂程度显著高于老龄鼠（Ｐ＜０．０１）。     

Mapping of Sequence-Tagged Sites in Rice by Single Strand Conformation Polymorphism

The conditions for efficient single-strand conformation polymorphism(SSCP) detection were examined for its application to mappingof DNA regions in the rice genome. Temperature for electrophoresisand glycerol concentrations in gel affected SSCP patterns significantly.The optimal detection conditions for SSCP also depends on thenucleotide sequences of fragments analyzed. Fragments over 300bp show complicated patterns depending on their nucleotide sequencesand were not suitable for SSCP analysis. Seventy primer pairswere designed from the sequence data available to amplify DNAregions as sequence tagged sites (STSs), and 39 of these STSswere found to generate SSCP between japonica rice (Nipponbare)and indica rice (Kasalath) in at least one of the experimentalconditions. The maps of DNA fragments amplified from 186 F₂-plantDNAs with 17 primer pairs were successfully determined. Thisdirect mapping method of the amplified DNA fragments with PCRis simple and quite sensitive, and can be used to set markersin the gap regions of a genetic linkage map.     

用脉冲电场凝胶电泳检测电离辐射所致哺乳动物细胞DNA双链断裂

 本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。