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。     

一种简单高效的5'-突出末端平端化方法

目的:报道一种利用Pfu DNA聚合酶延伸法补平限制片段5’-突出末端的平端化方法。方法:Pfu DNA聚合酶是用于PCR扩增的常规高保真DNA聚合酶,可在DNA模板和dNTPs存在条件下,沿5’→3’方向催化寡聚核苷酸聚合。由于终产物为平末端,可利用这一特点进行限制片段5’-突出末端补平。本文采用这一方法消除pAN7-1潮霉素抗性标记末端的XbaⅠ位点,以便载体构建中能够利用这一常见位点。pAN7-1先用XbaⅠ酶切成线性化载体,产生的5’-突出末端再用Pfu DNA聚合酶延伸法补平,通过平端化载体自连后XbaⅠ位点的消除来评价Pfu DNA聚合酶的平端化效果。结果:随机挑取3个重组子提取质粒均不能再用XbaⅠ切开,表明XbaⅠ位点成功消除。结论:Pfu DNA聚合酶具有高效率及高保真特性,因此本法简单高效而且经济适用。     

利用染色质免疫共沉淀技术确定转录因子RIN调控的靶基因

以粉红期番茄果实为材料,用含不同浓度甲醛的缓冲液交联DNA和蛋白质,利用超声波将其染色质随机断裂成大小为200-1 000 bp的片段,用RIN蛋白的特异性抗体免疫沉淀与RIN蛋白结合的DNA片段,然后解交联和纯化DNA片段,最终用普通PCR试验和测序验证与转录因子RIN结合的DNA序列.结果表明,适用于番茄果实的最佳ChIP试验条件为:用1％甲醛溶液交联DNA和蛋白质的复合物;用20％功率,工作6s,间隔10s,脉冲3次超声破碎该复合物,可以得到适当大小的片段,用于后续的试验.普通PCR和测序验证结果证明转录因子RIN与LeACS2和LeACS4启动子区域的CArG box序列结合.     

猪基质Gla蛋白基因序列多态及生物信息学分析

选择具有明显品种差异的3个猪品种(可乐猪、白香猪和大约克)构建品种DNA池,采用PCR产物直接测序法研究公猪MGP基因5’侧翼调控区、外显子1共1 112 bp范围内可能与精液品质相关的序列多态性,快速筛查到两个SNPs(C-849T、C-895T).进一步利用生物信息学分析MGP基因5’侧翼调控序列.预测出评分为95分以上的转录因子结合位点有31个,发现了CpG岛.C-849T和C-895T的变异则使白香猪比可乐猪和大约克多出了两个HSF可能的结合位点(85.9分).1个TATA可能的结合住点(91.4分),1个cdxA可能的结合位点(92.9分).研究结果为进一步研究MGP转录效率以及对猪精液品质的影响奠定基础.     

基于锚定PCR技术对10种重要农业害虫微卫星DNA位点的筛选及其特征分析

微卫星DNA广泛存在于真核和原核生物的基因组中,具有多态性丰富、易于检测等特点,在遗传图谱的构建、动植物遗传学育种等方面被广泛应用。利用5′锚定PCR技术对桃小食心虫、桃蛀螟、玉米螟、二点委夜蛾、花蓟马、黄胸蓟马、棕榈蓟马、斑翅果蝇、稻水象甲、扶桑绵粉蚧10种重要农业害虫进行微卫星DNA筛选,并分析各个物种的微卫星DNA的特点。不同物种的阳性克隆率、微卫星比率、克隆效率和冗余率存在不同程度的差异;在核苷酸碱基数目上,主要为二核苷酸重复序列,占重复位点总数的93．2％～100％,三、四核苷酸重复位点较少。在二核苷酸重复位点中, CA/TG重复位点最为丰富,占重复位点总数的89．2％～100％。这与使用的锚定引物密切相关;10种害虫的微卫星DNA平均重复次数为6．7～8．9次,其中玉米螟具有最高的微卫星DNA重复次数（34次）;在序列类型上,完全型序列占上述害虫序列总数的91．0％～100％。5′锚定PCR技术能够快速挖掘害虫的微卫星DNA位点,本研究结果为这些害虫微卫星位点的进一步利用奠定了基础。     

一种简单高效的5＇-突出末端平端化方法

目的：报道一种利用Pfu DNA聚合酶延伸法补平限制片段5’-突出末端的平端化方法。方法：Pfu DNA聚合酶是用于PCR扩增的常规高保真DNA聚合酶,可在DNA模板和dNTPs存在条件下,沿5’→3’方向催化寡聚核苷酸聚合。由于终产物为平末端,可利用这一特点进行限制片段5’-突出末端补平。本文采用这一方法消除pAN7-1潮霉素抗性标记末端的XbaⅠ位点,以便载体构建中能够利用这一常见位点。pAN7-1先用XbaⅠ酶切成线性化载体,产生的5’-突出末端再用Pfu DNA聚合酶延伸法补平,通过平端化载体自连后XbaⅠ位点的消除来评价Pfu DNA聚合酶的平端化效果。结果：随机挑取3个重组子提取质粒均不能再用XbaⅠ切开,表明XbaⅠ位点成功消除。结论：Pfu DNA聚合酶具有高效率及高保真特性,因此本法简单高效而且经济适用。     

碳离子束辐照对质粒DNA的损伤效应

为了证明DNA双链断裂(DSB)片段分布与DNA序列有关的假设,采用32keV／μm的^12C^6 离子和45ke V／μm的^13C^6 离子分别辐照pUCl8质粒,结合限制性内切酶处理,进行琼脂糖凝胶电泳,分析DNA断裂和片段分布。结果表明,除了由一个DSB导致的线性DNA带外,还出现了一条新的、小分子量线性DNA带;限制性内切酶处理后,有另一条线性DNA带产生。证明重离子辐照诱导的DSB是非随机分布的,DNA分子上存在对电离辐射相对敏感的位点。     

果蝇程序化死亡基因5(PDCD5)同源cDNA的克隆和序列分析

 为了解人类白血病细胞凋亡相关新基因 TFAR1 9(PDCD5,programmed cell death5)在不同种属间的序列同源性 ,利用 EST(expression sequence tag)拼接、RT- PCR、DNA序列测定技术及计算机分析技术 ,首次成功地进行了果蝇 PDCD5同源 c DNA编码区基因克隆和序列分析 .发现果蝇与小鼠及果蝇与人 PDCD5在核苷酸水平上分别有 57.5%和 57.1 %的同源性 ,在氨基酸水平上分别有 46.8%和 46.4%的同源性 .功能区分析发现 ,果蝇 PDCD5c DNA编码 1 33个氨基酸 ,计算机预测可能是一种核蛋白 ,含 5个可能的酪蛋白激酶 (casein kinase )磷酸化位点 ,2个可能的 PKC磷酸化位点 ,与人 PDCD5的功能区类似 .因而果蝇 PDCD5是与人 PDCD5同源的新基因 ,可能都与细胞程序化死亡相关 .     

血管内皮生长因子基因3'UTR 不同基因型载体的构建与鉴定

目的:构建含SNP位点的血管内皮生长因子(VEGF)基因3’UTR的荧光素酶报告基因载体,为进一步揭示VEGF基因3’UTR的单核苷酸多态性(SNP)影响肺癌发病风险的分子机制奠定基础。方法:以rs3025039和rs3025040两个位点均为C纯合子的非癌症病人血液DNA为模板,扩增出两位点为C/C单体型、长度为1448 bp的VEGF基因3’UTR目的片段,测序验证后将其克隆至pMIR-REPORT荧光素酶报告基因载体上,得到重组质粒pMIR-C/C。同时,我们以pMIR-C/C为模板定点突变两个SNP位点,得到具有T/T单体型的重组质粒pMIR-T/T。将各重组质粒转化大肠杆菌DH10B,筛选阳性克隆后提取质粒进行双酶切鉴定及DNA测序鉴定。结果:单菌落质粒测序验证显示带有C/C单体型的VEGF基因3’UTR重组质粒pMIR-C/C构建成功;经两次定点突变,成功将pMIR-C/C质粒转变为pMIR-T/T,经测序验证未引入任何其他突变。同时生物信息学预测还显示rs3025040位点位于miR-199a/b与VEGF基因mRNA的结合位置,其改变可以影响miRNA与mRNA的结合效率。结论:本研究成功构建了含有两个连锁SNP的VEGF基因3’UTR的荧光素酶报告基因载体,为今后VEGF基因3’UTR的功能研究奠定基础。     

黑曲霉糖化酶高产和低产菌株糖化酶基因调控区的克隆及其分析比较

以PCR合成的糖化酶高产菌株黑曲霉(Asp. Niger)T21糖化酶基因5’近端非编码区588bp(EcoRI-BamHI)的序列为探针,从T21染色体DNA中克隆到近2.0kb的糖化酶基因5’端非编码区序列,并以此序列为探针从糖化酶低产菌株黑曲霉3.795(T21的诱变出发株)的染色体DNA中克隆到1.5kb的糖化酶基因5’端非编码区序列。该二序列的分析测定结果表明,其结构特征与文献报道的黑曲霉糖化酶基因5’端非编码区的基本一致,被称为“核心启动子”(Core promoter)的TATAAAT框及GCAAT框,分别在翻译起始点的-109bp及-178bp处。此外,在曲霉amdS,amyB基因中已发现有调控功能的CCAAT序列存在于-449bp和-799bp处。高产和低产菌株糖化酶基因5’端非编码区序列的分析比较结果表明,有9个部位的碱基发生了变化。此实验结果为进一步研究黑曲霉糖化酶基因在转录水平上的调控规律打下了基础。     

Specificity of DNA cleavage by ultrasound

Cleavage of double-stranded DNA fragments with known nucleotide sequences upon sonication at 22 and 44 kHz was studied by PAGE. The cleavage rate was shown to depend on the fragment size, pH, ionic strength, and temperature. Double-strand breaks occurred preferentially in 5′-CpG-3′ dinucleotides. The strand was broken between C and G so that the phosphate group was at the 5′ side of G in the products. The cleavage rate proved to depend on the sequences flanking the cleavage site. The character of cleavage changed in the presence of Pt-bis-netropsin, a sequence-specific ligand that alters the local conformation of DNA.     

利用优化的5′-RACE实验定位副溶血弧菌基因的转录起始位点

目的:优化5′-cDNA末端快速扩增(5′-RACE)实验平台,用于定位副溶血弧菌(VP)基因的转录起始位点。方法:提取VP的总RNA,用rDNaseⅠ消化去除可能污染的基因组DNA;利用T4 RNA连接酶将已知序列的寡核苷酸片段连接至RNA的5′端,进而将其逆转录成cDNA;以cDNA为模板,采用巢式PCR技术扩增目的基因DNA片段,并将其直接克隆入T载体;最后通过测序比对的方法确定靶基因的转录起始位点。利用引物延伸实验进一步研究VPA1027的转录起始位点,以检验5′-RACE实验结果的可靠性。结果:5′-RACE实验结果表明,VPA1027、scrG、scrA、cpsA及VPA0198的转录起始位点分别为G(-103)、G(-70)、T(-205)、C(-129)和G(-238)(翻译起始位点为+1);引物延伸结果显示,VPA1027的转录起始位点也为G(-103)。结论:优化后的5′-RACE实验可以精确定位VP基因的转录起始位点。     

Sequence-specific ultrasonic cleavage of DNA

We investigated the phenomenon of ultrasonic cleavage of DNA by analyzing a large set of cleavage patterns of DNA restriction fragments using polyacrylamide gel electrophoresis. The cleavage intensity of individual phosphodiester bonds was found to depend on the nucleotide sequence and the position of the bond with respect to the ends of the fragment. The relative intensities of cleavage of the central phosphodiester bond in 16 dinucleotides and 256 tetranucleotides were determined by multivariate statistical analysis. We observed a remarkable enhancement of the mean values of the relative intensities of cleavage (cleavage rates) in phosphodiester bonds following deoxycytidine, which diminished in the row of dinucleotides: d(CpG) > d(CpA) > d(CpT) >> d(CpC). The cleavage rates for all pairs of complementary dinucleotides were significantly different from each other. The effect of flanking nucleotides in tetranucleotides on cleavage rates of all 16 types of central dinucleotides was also statistically significant. The sequence-dependent ultrasonic cleavage rates of dinucleotides are consistent with reported data on the intensity of the conformational motion of their 5′-deoxyribose. As a measure of local conformational dynamics, cleavage rates may be useful for characterizing functional regions of the genome.     

The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells

The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5′-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5′-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5′-GT*A and 5′- TGT* trinucleotide sequences, and 5′-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5′-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine–pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the ?3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.     

Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA.

Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.     

血管内皮生长因子基因3＇UTR不同基因型载体的构建与鉴定

目的：构建含SNP位点的血管内皮生长因子（VEGF）基因3＇UTR的荧光素酶报告基因载体,为进一步揭示VEGF基因3＇UTR的单核苷酸多态性（SNP）影响肺癌发病风险的分子机制奠定基础。方法：以rs3025039和rs3025040两个位点均为C纯合子的非癌症病人血液DNA为模板,扩增出两位点为C/C单体型、长度为1448 bp的VEGF基因3＇UTR目的片段,测序验证后将其克隆至pMIR-REPORT荧光素酶报告基因载体上,得到重组质粒pMIR-C/C。同时,我们以pMIR-C/C为模板定点突变两个SNP位点,得到具有T/T单体型的重组质粒pMIR-T/T。将各重组质粒转化大肠杆菌DH10B,筛选阳性克隆后提取质粒进行双酶切鉴定及DNA测序鉴定。结果：单菌落质粒测序验证显示带有C/C单体型的VEGF基因3＇UTR重组质粒pMIR-C/C构建成功;经两次定点突变,成功将pMIR-C/C质粒转变为pMIR-T/T,经测序验证未引入任何其他突变。同时生物信息学预测还显示rs3025040位点位于miR-199a/b与VEGF基因mRNA的结合位置,其改变可以影响miRNA与mRNA的结合效率。结论：本研究成功构建了含有两个连锁SNP的VEGF基因3＇UTR的荧光素酶报告基因载体,为今后VEGF基因3＇UTR的功能研究奠定基础。     

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.     

用支持向量机预测人类基因5’／3’选择性剪切位点

选择性剪切是调解基因表达的重要机制.识别选择性剪切位点是后基因组时代的一个重要工作.本文从最新的EBI人类基因选择性剪切数据库中,选取5＇/3＇选择性剪切位点作为正集,选取在剪切位点附近的假剪切位点作为负集,并把所有的选择性剪切位点和假剪切位点随机分成训练集和测试集.本文选用的预测选择性剪切位点的方法是基于位置权重矩阵和离散增量的支持向量机方法.此方法仅基于训练集,以不同位点的单碱基概率和序列片断的三联体频数作为信息参数,利用位置权重矩阵和离散增量算法结合支持向量机,得到了选择性供体位点和受体位点的分类器,并用此分类器对测试集中的选择性供体位点和受体位点进行预测.对独立测试集中的选择性供体位点和选择性受体位点的预测成功率分别为88.74%和90.86%,特异性分别为85.62%和81.19%.本文预测选择性剪切位点的方法成功率高于其它选择性剪切位点预测方法预测成功率,此预测方法进一步提高了对选择性剪切位点的理论预测能力.     

猪α-1,3-半乳糖转移酶基因打靶载体的构建

目的:构建猪α-1,3-半乳糖转移酶(GGTA1)基因的正负筛选打靶载体。方法:以原代猪胚胎成纤维细胞基因组DNA为模板,采用长程PCR方法扩增出GGTA1基因的2条片段;以长约2kb包含部分第9外显子的片段为同源短臂,在XbaⅠ和ClaⅠ位点插入pLoxP质粒正筛选标记neo基因的3'端;以长约5.4kb包括部分第8外显子、全部第8内含子及部分第9外显子的片段为同源长臂,于NotⅠ位点插入该质粒中neo基因的5'端;2.7kb的负筛选标记tk基因位于载体中同源短臂的3'端外侧。结果与结论:酶切、PCR及测序结果表明,同源臂被正确连接至质粒pLoxP,成功构建了猪GGTA1基因正负筛选打靶载体pSL/GT。     

β淀粉样肽单克隆抗体可变区基因的5′RACE扩增及序列分析

目的:克隆并分析抗β淀粉样肽单克隆抗体轻链与重链可变区基因。方法:从分泌抗β淀粉样肽单克隆抗体的杂交瘤细胞株A8中提取总RNA,根据恒定区序列设计基因特异性引物,通过5′RACE法扩增抗体的轻链和重链可变区基因,测定并分析可变区基因序列,并克隆入pMD18-T载体。结果:重链可变区基因序列全长450bp,编码150个氨基酸残基;轻链可变区基因序列全长429bp,编码143个氨基酸残基。在GeneBank中对氨基酸序列进行比对分析,二者均符合小鼠IgG可变区基因的特征。根据Kabat法则对A8抗体轻链和重链可变区氨基酸序列基因进行分析并确定了3个抗原互补决定区(CDR)、4个框架区(FR)和信号肽。结论:通过5′RACE法得到了抗β淀粉样肽单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构,以及对该抗体进行人源化改造奠定了基础。