一个α,β地中海贫血复合家系β珠蛋白基因的进一步研究

 前文~[1]曾报道广西一个α,β地中海贫血复合家系的血红蛋白组成及α珠蛋白基因分析结果,并讨论了各成员可能的β珠蛋白基因结构情况。本文利用先进的PCR即基因扩增技术,结合特异寡核苷酸探针斑点杂交及扩增后直接测定DNA序列的技术,进一步研究并彻底搞请了该家系各成员的β珠蛋白基因结构情况。结果显示:母亲及两个弟弟都是编码子41—42TTCT四个碱基缺失造成框架位移所致β地中海贫血的杂合子。父亲与先证者的β基因均属正常。前三个成员均为α地贫复合β地贫,其α与β珠蛋白链合成的不均衡状态得到改善,贫血症状也明显轻。     

克隆化反向杂交探针的制备

反向斑点杂交是将寡核苷酸探针固定在膜上,用标记的靶序列与固定在膜上的探针进行杂交．与正向杂交相比,它通过一次杂交即可确定多种基因型,是一种快速筛查DNA点突变的诊断方法.我们选择β－地中海贫血基因-28 (A→ G), CD17 (A→T) and CD41～42 (-TTCT) 三种点突变为模型采用PCR扩增产生串联多拷贝序列探针并将其克隆化．将克隆化探针固定于尼龙膜上,与同位素标记的β－珠蛋白基因PCR片段进行杂交,检测β－地贫患者的基因型．     

2型腺相关病毒载体介导正常人β珠蛋白基因在重型地贫流产胎儿造血细胞中的表达

目的:探讨2型重组腺相关病毒(recombinant adeno-associated virus 2,rAAV2)载体介导人β-珠蛋白基因转染地贫患者造血细胞治疗β-地中海贫血的可行性.方法:分离β 41-42/β 654杂合子型重型β-地贫流产胎儿造血细胞,经rAAV2-β-globin病毒转染(MOI=50)后移植入经X射线照射的BALB/c裸小鼠体内,分别于移植后14d、21d处死受体小鼠,RT-PCR及等位基因特异性PCR法检测人珠蛋白基因在受体小鼠体内的表达.结果:RT-PCR方法于3只受体小鼠骨髓样本中成功检测到人β-actin和人β-珠蛋白基因的表达,而21d处死的转染与对照小鼠外周血样本中未检测到人β-珠蛋白基因表达;等位基因特异性PCR方法在所有受体鼠体内同时检测到β 41-42和β 654突变基因,以及正常β-珠蛋白基因的表达,但rAAV2-β-globin转染组小鼠体内正常人β-珠蛋白基因表达水平明显高于对照组.结论:rAAV2可有效转染β 41-42/β 654杂合子型重型地中海贫血患者造血细胞,并通过exvivo途径介导β-珠蛋白转基因的体内表达,提高红系细胞内正常β-珠蛋白基因的表达水平.     

3′-末端标记法制备乙型肝炎病毒(HBV)直接重复序列(DR)的生物素化的寡核苷酸探针

本文首次报告用3’—末端核苷酸转移酶将生物素—11—dUTP标记到人工合成的21寡核苷酸上,该d(NMP)_(21)与HBV DNA长链缺口区附近的序列互补,并含HBV DNA的直接重复序列(Direct Repeat)。本文对其标记条件、检出方法进行观察比较,找出合适的条件,制成的生物素化d(NMP)_(21)探针可与标准的HBVDNA进行杂交,检测敏感度为25pg。用这种探针可以从乙型肝炎病人血清中检出HBV DNA。     

一种基于寡核苷酸微阵列芯片的多重可扩增探针杂交技术

多重可扩增探针杂交技术(multiplex amplifiable probe hybridization,MAPH)是近年来发展起来的一种用于基因组中DNA拷贝数检测的新技术。并发展了一种基于寡核苷酸微阵列芯片的MAPH技术。该方法根据所检测的DNA序列,制备若干具有通用引物的FCR产物作为可扩增探针组,与固定在尼龙膜上待测的基因组DNA杂交。用磁珠回收特异性杂交的探针,经生物素标记的通用引物扩增后,与相应的寡核苷酸微阵列芯片杂交。该特异性的寡核苷酸微阵列芯片包括10个抗肌营养不良基因的外显子探针和阴性、阳性探针。杂交清冼后,链霉亲和素-Cy3染色用芯片扫描仪得到杂交的荧光图像。分析荧光信号的强度差异给出特定基因片段拷贝数的变化。该方法用微阵列技术代替MAPH中的电泳检测技术,可大幅度增加检测的通量。选择了一个正常男性、一个正常女性和一个肌营养不良症患者的基因组DNA来进行验证。结果表明,该方法能够同时给出抗肌营养不良基因多个外显子中的基因片段拷贝数差异信息。     

用合成的寡核苷酸探针鉴定中国人β-地中海贫血基因突变

用人工合成的六种对β-地中海贫血基因特异的寡核苷酸作为杂交探针,对10例β-地中海贫血病人及其父母的β-珠蛋白基因进行分析,鉴定出六种β-地中海贫血突变:(1)TATA Box-28 A→G;(2)IVS-1n.5 G→C;(3)Codon 17 A→T;(4)codons 41—42—46p;(5)Codons 71—72+A;(6)IVS-2 n.654C→T。本文分析的中国人β-地中海贫血患者中,上述六种突变所占的百分比分别为5％,10％,10％,40％,20％和15％。     

Hb D Punjab基因的鉴定——DNA 扩增在异常血红蛋白研究中的应用

本文应用作者设计的寡核苷酸引物和探针,通过聚合酶链反应(PCR)扩增β珠蛋白DNA序列,并通过限制酶EcoRI酶谱分析或寡核苷酸杂交,快速鉴定Hb D Punjab基因。应用这种技术先后对汉、藏、哈萨克等3个民族4个家系的Hb D Punjab基因进行了鉴定。     

用直接测定基因组DNA序列的方法检测β-地中海贫血基因突变

本文报道一种结合聚合酶链反应(PCR)技术直接测定基因组DNA中单考贝基因片段序列的方法,以及利用这种方法测定两例β-地贫纯合子的β珠蛋白基因序到结果。测定出基因点突变,一例为编码子17(A→T)突变纯合子,另一例为编码子69(G→A)突变纯合子。针对上述两个点突变合成寡核苷酸片段,末端标记~(82)P后为探针进行斑点杂交的结果与测序结果一致。     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

促甲状腺激素释放激素受体mRNA在大鼠睾丸中的表达(英文)

本应用原位杂交技术在大鼠睾丸恒冷箱切片上研究了促甲状腺激素释放激素受体（TRH－R）RNA的表达和定位。由DNA合成仪合成两个含48个碱基的寡核苷酸探针,两个探针分别与小鼠垂体TRH－R1005－1052和1332－1379区段的cDNA互补,生物素在5′末端标记寡核苷酸探针。结果显示TRH－R寡核苷酸探针与其互补的mRNA杂交信号集中在大鼠睾丸的间质细胞中,生精细胞地交信号,杂交信号的强度依探针浓度而增加,该结果表明RTH可能通过自分泌调节生殖功能和发育,TRH－R作用途径可能与在垂体的作用类似。     

Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients

Parasite DNA amplified by PCR from blood of 73 chagasic children and adults of two endemic areas of Chile were studied by Southern blot and/or dot blot hybridization analysis with a panel of three minicircle probes corresponding to the parasite genotypes (clonets 19, 33 and 39). The hybridization pattern of the PCR positive samples identified clonets 39, 19/20, and 32/33 with frequencies of 0.84, 0.32 and 0.26, respectively. A total of 31 samples corresponded to mixed infections. The most frequently found mixtures were: clonets 39 and 19/20 (14 cases), followed by clonets 39 and 32/33 (8 cases), clonets 39, 32/33 and 19/20 (8 cases), and clonets 32/33 and 19/20 (1 case). Amplified DNA from 9 cases showed no hybridization signal with none of the three studied probes indicating that other genotypes different to the ones mentioned are circulating in humans, but that the clonets used as probes are the most prevalent ones in terms of transmission in the endemic areas studied. A biological characterization of 34 T. cruzi populations isolated from the xenodiagnosis of the patients was performed on an experimental murine model. The biochemical characterization of the parasite populations by molecular karyotype determined that the most frequent parasite isolated from patients belongs to clonet 39.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil.

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

A DNA probe for specific detection of Mycoplasma pulmonis

Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.     

Combined subtraction hybridization and polymerase chain reaction amplification procedure for isolation of strain-specific Rhizobium DNA sequences.

A novel subtraction hybridization procedure, incorporating a combination of four separation strategies, was developed to isolate unique DNA sequences from a strain of Rhizobium leguminosarum bv. trifolii. Sau3A-digested DNA from this strain, i.e., the probe strain, was ligated to a linker and hybridized in solution with an excess of pooled subtracter DNA from seven other strains of the same biovar which had been restricted, ligated to a different, biotinylated, subtracter-specific linker, and amplified by polymerase chain reaction to incorporate dUTP. Subtracter DNA and subtracter-probe hybrids were removed by phenol-chloroform extraction of a streptavidin-biotin-DNA complex. NENSORB chromatography of the sequences remaining in the aqueous layer captured biotinylated subtracter DNA which may have escaped removal by phenol-chloroform treatment. Any traces of contaminating subtracter DNA were removed by digestion with uracil DNA glycosylase. Finally, remaining sequences were amplified by polymerase chain reaction with a probe strain-specific primer, labelled with 32P, and tested for specificity in dot blot hybridizations against total genomic target DNA from each strain in the subtracter pool. Two rounds of subtraction-amplification were sufficient to remove cross-hybridizing sequences and to give a probe which hybridized only with homologous target DNA. The method is applicable to the isolation of DNA and RNA sequences from both procaryotic and eucaryotic cells.