一种用少量标本快速构建老年性白内障消减cDNA文库的方法

为从少量标本中获得含较多大片段的、高质量的老年性白内障消减cDNA文库,利用磁珠分离、生物素标记的改良消减杂交法获得差异cDNA,利用选择性PCR法扩增其中大片段差异cDNA,从而成功构建老年性白内障消减cDNA文库.在文库中随机挑取的22个克隆中,1 000 bp以上的片段有7个,占31.8%,750 bp以上有15个,占68.2%.所得cDNA片段较大,可以满足下一步研究需要.改良消减杂交法结合选择性PCR法可以从少量标本中快速有效地获得大片段高质量的消减cDNA文库.     

北柴胡细胞色素P450酶基因的克隆及其植物过量表达载体的构建

目的:克隆北柴胡中可能参与柴胡皂苷生物合成的细胞色素P450酶基因,构建其过量表达载体,为通过转基因验证其功能奠定基础。方法:在454高通量测序获得5'和3'端部分cDNA序列的基础上,利用LD-PCR方法获得全长cDNA,根据全长cDNA序列设计含有酶切位点的PCR引物,利用高保真酶,以RNA反转录产物为模板PCR扩增细胞色素P450酶基因的开放读框,扩增产物与pEASY-T1 Simple载体连接,转化大肠杆菌DH5α;重组质粒pT1-P450经菌液PCR和酶切方法验证后测序,采用NCBI在线Blastx、DNAman和MEGA4软件对序列进行生物信息学分析,随后将pT1-P450的酶切产物插入双元载体pCAMBIA-SUPER 1300,菌液PCR和酶切验证重组质粒p1300-P450。结果:扩增到了北柴胡细胞色素P450酶基因BcCYP87E,构建了这一基因的过量表达载体。结论:细胞色素P450酶基因的克隆和转基因载体的构建,为后续开展转基因研究,验证其生物功能奠定了基础。     

甘菊cDNA-AFLP反应体系的优化

以甘菊为试验材料,研究了影响cDNA-AFLP反应体系的几个关键因素,建立了适宜甘菊的cDNA-AFLP分析体系,并得到了清晰可辨的cDNA-AFLP指纹图谱.结果表明:适用于甘菊叶片总RNA提取的方法为改进的Trizol法;酶切连接采用一步法,dscDNA酶切用量为300 ng,酶切连接时间为8 h;PCR选择性扩增反应时,反应体系中最佳组合为:引物浓度0.4 mM、Mg2+浓度1.25 mM、Taq酶浓度0.9 U、dNTP浓度0.3 mM.     

应用限制性显示PCR技术构建细菌poly(A)化mRNA的cDNA文库

针对细菌mRNA poly(A)化位点的高度多态性,利用oligo(dT)与poly(A)特异结合的特性,以oligo(dT)一纤维素纯化mRNA,并以oligo(dT)18为引物逆转录合成cDNA,用限制性内切酶消化cDNA,所得的限制性内切酶片段与通用接头相连,通过10个选择性引物组合进行选择性PCR,使各片段得以扩增并分布于10个亚组中,并进行克隆,成功地克隆了100多个基因片段,已对其中40个进行了测序分析,探讨了限制性显示PCR技术在细菌poly(A)化mRNA cDNA库构建中的应用价值。     

Cloning of differentially expressed genes in highly and low metastatic rat osteosarcomas by a modified cDNA-AFLP method.

To identify differentially expressed genes between highly and low metastatic rat transplantable osteosarcomas, we applied a modified AFLP (amplified fragment length polymorphisms) method for cDNA subtraction. The specific point of our modification is selective amplification using suppression PCR technique after restriction enzyme cutting. Our cDNA-AFLP gave high reproducibility (about 95%) in mRNA patterns and enabled us to clone four dominantly expressed genes in a highly metastatic tumor line. Three showed homology with known genes, encoding Ki-67, a proliferation-associated effective marker of malignancy, type IV collagen alpha-3, a major component of basement membrane, and KIAA77 for which the function is unknown. Although one fragment showed no database homology, we revealed a derivation from the rat homologue of the Drosophila melanogaster diaphanous gene (Dia) by cloning of longer cDNA. Dia genes, known to affect actin filament formation, are downstream effectors of Rho small GTPase. The results suggest that alterations in the expression of cytoskeletal protein, basement membrane elements, and proliferative markers may be important for metastasis of osteosarcomas.     

Development of a microarray assay that measures hybridization stoichiometry in moles

Microarray data is most useful when it can be compared with other genetic detection technologies. In this report, we designed a microarray assay format that transforms raw data into a defined scientific unit (i.e., moles) by measuring the amount of array feature present and the cDNA sequence hybridized. This study profiles a mouse reference universal RNA sample on a microarray consisting of PCR products. In measuring array features, a labeled DNA sequence was designed that hybridizes to a conserved sequence that is present in every array feature. To measure the amount of cDNA sample hybridized, the RNA sample was processed to ensure consistent dye to DNA ratio for every labeled target cDNA molecule, using labeled branched dendrimers rather than by incorporation. A dye printing assay was then performed in order to correlate molecules of cyanine dye to signal intensity. We demonstrate that by using this microarray assay design, raw data can be transformed into defined scientific units, which will facilitate interpretation of other experiments, such as data deposited at the Gene Expression Omnibus and ArrayExpress.     

Efficient procedure for site-directed mutagenesis mediated by PCR insertion of a novel restriction site

Better understanding of proteins'' structure/function relationship and dissecting their functional domains are still challenges yet to be mastered. Site-directed mutagenesis approaches that can alter bases at precise positions on the gene sequence can help to reach this goal. This article describes an efficient strategy that can be applied not only for both deletion and substitution of target amino acids, but also for insertion of point mutations in promoter regions to study cis-regulating elements. This method takes advantage of the plasticity of the genetic code and the use of compatible restriction sites.Key words: site-directed mutagenesis, restriction site, cloning, PCRUnderstanding the proteins structure/function relationship and dissecting their functional domains is one of the biggest challenges to current proteomic studies.¹ This is mainly achieved by site-directed mutagenesis experiments that can alter bases at precise positions on the gene sequence.² Modifying DNA sequences has become feasible with PCR amplification.³ During the last decade, several strategies have been developed to simplify this approach and increase its efficiency.⁴ The introduction of a site-directed mutation can be realized by one or more PCR reactions. Most of the strategies used in site-directed mutagenesis are based on a substitution of a single base, which leads to a change in one amino acid. This article describes an efficient strategy that can be applied for either deletion or substitution of target amino acids. This strategy is based on performing PCR reactions to create a new restriction site in the sequence of origin, corresponding to the desired mutation. The choice of the restriction site to be created depends on the nature of the amino acid that one desires to introduce in the protein sequence. Since such restriction sites may extend beyond the mutated codon. The preservation of the other codon is done by taking advantage of the plasticity of the genetic code where one amino acid can be encoded by multiple codons.This method was performed in two steps (Fig. 1). In the first step, the DNA sequence of interest, cloned in a plasmid, served as a template for two PCR reactions. Two PCR products are generated. The first one consists of the beginning of the sequence, from the start codon to the mutagenized amino acid codon, where the forward primer bears the start codon region and the reverse primer bears the newly introduced restriction site at the same location of the mutagenized codon. The second PCR product consists of the end of the coding sequence, from the mutagenized amino acid codon to the stop codon. This fragment is generated using a forward primer bearing the same new restriction site as the first PCR product''s reverse primer, and a reverse primer bearing the stop codon region. The two PCR products were cloned separately into a vector in the appropriate orientation. In the second step, the cloning vector bearing the first PCR product was digested with a restriction enzyme site in the vector, and by the restriction enzyme corresponding to the restriction site created by the reverse primer used in the PCR reaction. The resulting fragment was cloned into the vector containing the second PCR fragment, predigested with same two restriction enzymes. The whole mutagenized coding sequence is reassembled by in-frame subcloning of the 3′ end of the coding sequence downstream the 5′ end. All the PCR products were generated using the high fidelity Pfu DNA Polymerase (Promega, Madison, WI USA). For any site-directed mutagenesis experiment, this two-step cloning procedure requires the use of appropriate PCR primers that harbor the desired mutation of the target amino acid. These primers are partially overlapping and contain a common or complementary restriction site enabling the in-frame assembly of the whole coding sequence.Open in a separate windowFigure 1Mutagenesis strategy by restriction enzyme site insertion. (A) In the first step, two PCR products were generated using the full length coding sequence as template. The mutation is carried by the two primers b and c, which are flanked by the same or compatible restriction enzyme''s site (white segment). Both PCR products are separately cloned in the cloning vector in the appropriate orientation. In the second step, the whole mutagenized coding sequence is reassembled by in-frame sub cloning of the 3′ end of the coding sequence downstream the 5′ end. (B) Substitution of threonine by arginine as a result of the insertion of a BglII restriction site. DNA sequencing is carried out to make sure that only the desired change is introduced in the coding sequence. (B-1) The sequence of the native cDNA. (B-2) the sequence of the mutagenized cDNA included BglII restriction site sequence.This approach has been used in a recent study to address the structure/function relationship of the STAS domain of the Arabidopsis thaliana Sultr1;2 sulfate transporter.⁵ A good example of this approach is the replacement of the threonine-serine couple at position 587–588 with an arginine-serine couple. The codon for threonine is: TGT, and that for arginine is: TCT. Serine can be encoded by both TCA and AGA codons. The chosen restriction site used for the reassembly of the whole coding sequence is that of the BglII enzyme: TCT AGA. The insertion of this restriction site enables the substitution of the Thr in position 587 with an Arg while preserving the serine residue in position 588. The BglII restriction site is introduced in the reverse primer and the forward primer used to generate the first and second PCR products respectively. The DNA sequence of the reassembled mutagenized cDNA was checked by sequencing. Than it was expressed, under pGAL1O promoter bearing by pYES2 vector, in yeast mutant deficient in sulfate transporter and the mutagenic protein was detected by imunodetection.Bioinformatic study reveals that this method can be applied to checked a large number of substitutions, insertions or deletions and that finding the right restriction site is not a limiting factor (data no shown).In conclusion, this article describes an efficient two-step procedure for site-directed mutagenesis using primers bearing a restriction site, which is absent from the sequence of origin. The primers flanked by sequences introducing the same or compatible restriction sites mediate the incorporation of the mutation at the selection site. The choice of the restriction site depends on the nature of the desired mutation: insertion, substitution or deletion of an amino acid in a particular position. This strategy can be also used to insert point mutations in promoter regions to study cis-regulating elements.     

家蚕基因特异性CAPs标记获得及其分子系统学应用

选取家蚕attacin和alpha-amylase基因序列,设计特异性引物,在家蚕品系P50、C108和子一代 (F1) 中扩增。分别采用4种不同的限制性内切酶对扩增产物酶切,最后每个基因都获得了一个CAPs分子标记。依据所得的两个CAPs分子标记对12个品系的家蚕遗传多样性进行了初步研究,构建了其分子系统树。     

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli.

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5 F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHARdehydroascorbatereductase基因的cDNA与DNA全长序列,命名为BoDHAR。并利用双链接头介导PCR的染色体步行技术(genomewalking)克隆了其上游644bp的5′端序列。所获的BoDHAR基因全长1486bp,存在两个内含子,DNA编码区序列633bp,编码210个氨基酸;序列分析表明BoDHAR与同源基因AT1G19570.1cDNA序列有82.3%的一致性,推导的氨基酸序列有79.6%的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明BoDHAR在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。