Preliminary study on the detection of the SARS-CoV specific target cDNA fragments by multiplex PCR

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The re~sults indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.     

毛冠鹿ZFY、ZFX基因片段的克隆与性别鉴定

根据人和鼠性别分化相关的ZFY、ZFX基因序列设计引物,以雌雄毛冠鹿的基因组DNA为模板进行PCR扩增,将扩增产物克隆到pMD18T上,获得ZFY、ZFX重组克隆,并测定了ZFY、ZFX基因片段的序列,序列比较显示两者同源性达 91%,仅在少数位点有差异,以此确定AvaⅡ为ZFX上特异酶切位点,通过PCR扩增和AvaⅡ特异酶切对毛冠鹿性别进行鉴定。 Abstract: According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of AvaⅡ was found to be specific to ZFX gene. The results show that the combination of PCR with AvaⅡ digestion is a simple and sensitive way to identify the tufted deer sex.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

基因芯片技术检测肾综合征出血热病毒核酸的研究

To establish a rapid, sensitive and specific diagnostic assay for Hantavirus with microarray techniques, specific primers and probes were designed according to the conservative and specific DNA sequence of 76-118 strain and R22 strain. The probes were spotted on glass slides to form microarrays.The Cy3-1abled single stranded DNA fragments prepared by dissymmetical PCR were hybridized with the probes on the glass slides. The microarrays were scanned and analyzed with a scanner. The results showed that the DNA microarray could detect the different typed DNA of HTN and SEO with adequate specificity and sensitivity. The developed DNA microarray and techniques might be a very useful method for diagnosis and prevention, and could be widely applied in specific pathogens detection ofinfectious diseases such as hemorrhagic fever with renal syndrome.     

水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Xa-4紧密连锁的分子标记M55的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片DNA、半粒种子提取物及Xa-4基因的杂合体DNA的PCR特异扩增,初步建立了Xa-4的PCR标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Xa-4在这些材料中的分布情况。 Abstract　Based on the sequence of a DNA marker tightly linked to the rice bacterial blight（BB） resistance gene Xa-4, two primers were designated and synthesized to develop a PCR marker for the gene. Specific amplified polymorphism analysis was carried out with these primers on a set of BB resistance isogenic lines and pyramided lines developed by IRRI. Two PCR bands were revealed corresponding to lines with dominant Xa-4 and those with the recessive allele, respectively, regardless the lines pyramided with other resistance genes. A hybrid with heterozygous Xa-4 produced both of the two allele PCR pattern. Then, the PCR marker was used to survey a range of hybrid rice germplasm. The results of the germplasm survey will be useful in hybrid rice breeding programs aimed at exploiting Xa-4.     

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses （enJSRV）

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia （enJSRV-NM）, we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame （orf-x） that overlaps the 3＇ end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain （Accession No.AF153615） and USA JSRV21 strain （Accession No. AF105220）. The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.     

应用PCR技术对先天性长QT综合征KCNQ1 基因进行定点突变的研究

利用聚合酶链反应（PCR）技术对长QT综合征（LQTS）KCNQ1基因进行定点突变的研究。首先设计两对引物（包含预定的突变）,通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2-EGFP-KCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2-EGFP-KCNQ1基础上获得了KCNQ1 cDNA C934T的突变体,测序表明在序列中发生了预期的突变。将含突变点的pIRES2-EGFP-KCNQ1转染HEK293细胞后,在荧光显微镜下观察到被转染的HEK293细胞发出绿色荧光,表明含突变点的pIRES2-EGFP-KCNQ1得到了表达。Abstract: To study PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro. The site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR. Two sets of primers were designed according to the sequence of KCNQ1 cDNA, and mismatch was introduced into primers. Mutagenesis was performed in a three-step PCR. The amplified fragments from the third PCR which contained the mutation site were subcloned into the T-vecor PCR2.1.Then the fragments containing the mutation site was obtained from PCR2.1 with restriction enzyme digestion and was inserted into the same restriction site of pIRES2-EGFP-KCNQ1. With Effectene Transfection Reagent, pIRES2-EGFP-KCNQ1 was transfected into HEK293 cell. The sequencing analysis showed that the mutation site was correct. Mutation from T to C in 934 site of KCNQ1 cDNA was found. Under the fluorescence microscope, the green fluorescence was spread in the transfected HEK293 cell, meaning the pIRES2-EGFP-KCNQ1 containing the mutation site was expressed correctly.     

Candidate genes of hypertension with defective environmental expression

Previous studies in our laboratory have demonstrated that the thermosensitivity locus cosegregates with blood pressure and that the elevated expression and restriction fragment length polymorphism of HSP70 gene are associated with hypertension.Cell protection against environmental stressors such as heat and chemicals is often accompanied by up-regulated expression of a wide spectrum of heat shock genes(HSP).To further investigate the interrelation between HSP expression and blood pressure regulation,we employed an effective method of cloning 2 potential hypertension-related HSPs.Synthetic oligonucleotides corresponding either to a highly-conserved region of the known HSP family or a repetitive sequence in the proteinencoding gene were used as target primers for polymerase chain reaction(PCR).cDNA prepared from heat-stressed and non-stressed vascular smooth muscle cells(VSMC)of Brown Norway rats(BN.1x)and spontaneously hypertensive rats(SHRp) respectively served as template in the reaction.The PCR products were subsequently analyzed in a single-stranded conformational polymorphism(SSCP) electrophoresing system.Differential gene expression in BN.1x and SHRp was seen on autoradiographs of SSCP gel by comparing the migration patterns of PCR-amplified DNA fragments.Using this technique,we also found that HSP27 and a new member of the large HSP gene family were differentially expressed in BN.1x and SHRp VSMC.     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

Linkage mapping of starch branching enzyme III in rice (Oryza sativa L.) and prediction of location of orthologous genes in other grasses

 The chromosomal position of Starch Branching Enzyme III (SBEIII) was determined via linkage to RFLP markers on an existing molecular map of rice (Oryza sativa L.). A cDNA of 890 bp was generated using specific PCR primers designed from available SBEIII sequence data and used as a probe in Southern analysis. The SBEIII cDNA hybridized to multiple restriction fragments, but these fragments mapped to a single locus on rice chromosome 2, flanked by CDO718 and RG157. The detection of a multiple-copy hybridization pattern suggested the possibility of a tandemly duplicated gene at this locus. The map location of orthologous SBE genes in maize, wheat, and oat were predicted based on previously published genetic studies and comparative maps of the grass family. Received : 5 August 1996 / Accepted : 13 September 1996     

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。     

Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR

The structure of the 3' one-third of the dystrophin gene has not previously been established. We have used vectorette PCR on a yeast artificial chromosome containing part of the human dystrophin gene to determine that there are 20 exons in this region and to characterize adjacent intron sequences of each one. Combined with previous information on the remainder of the gene, this study shows that the coding sequence is distributed between 79 exons. We have used PCR between exons to measure the distances that separate the more closely clustered exons. Vectorette PCR products were used as probes on Southern blots to assign all the 3' exons to genomic HindIII fragments that are commonly detected in the analysis of dystrophin gene deletions. The results will be useful for determining the effect of genomic deletions on the translational reading frame, for setting up genomic PCR assays to confirm point mutations, for analyzing splice site mutations, and for investigating potential cis-acting elements involved in tissue-specific alternative splicing. Vectorette PCR using primers derived from cDNA sequence represents an efficient and widely applicable method for establishing gene structure and obtaining intron sequence flanking exons, starting from a genomic clone and a cDNA sequence.     

Cloning of cDNA encoding COMT from wheat which is differentially expressed in lodging-sensitive and -resistant cultivars

In the present study two cDNA fragments were cloned by nested-PCR using degenerate primers for COMT and found to be 93% identical at the nucleotide level. The deduced amino acid sequence of the two cDNAs showed a high degree of identity with COMT from other plants and were most similar to COMTs from monocots. RNA gel blot hybridization demonstrated that the wheat COMT gene W-cm5-1 was expressed in stem, root and leaf tissues. W-cm5-1 mRNA levels in the developing wheat stem were associated with the stem lodging character in two wheat cultivars. These results suggest that the action of the wheat COMT gene may be related to stem rigidity and lodging character in wheat.     

Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GSs) play an important role in determining the bread-making quality of bread wheat. However, LMW-GSs display high polymorphic protein complexes encoded by multiple genes, and elucidating the complex LMW-GS gene family in bread wheat remains challenging. In the present study, using conventional polymerase chain reaction (PCR) with conserved primers and high-resolution capillary electrophoresis, we developed a new molecular marker system for identifying LMW-GS gene family members. Based on sequence alignment of 13 LMW-GS genes previously identified in the Chinese bread wheat variety Xiaoyan 54 and other genes available in GenBank, PCR primers were developed and assigned to conserved sequences spanning the length polymorphism regions of LMW-GS genes. After PCR amplification, 17 DNA fragments in Xiaoyan 54 were detected using capillary electrophoresis. In total, 13 fragments were identical to previously identified LMW-GS genes, and the other 4 were derived from unique LMW-GS genes by sequencing. This marker system was also used to identify LMW-GS genes in Chinese Spring and its group 1 nulli–tetrasomic lines. Among the 17 detected DNA fragments, 4 were located on chromosome 1A, 5 on 1B, and 8 on 1D. The results suggest that this marker system is useful for large-scale identification of LMW-GS genes in bread wheat varieties, and for the selection of desirable LMW-GS genes to improve the bread-making quality in wheat molecular breeding programmes.     

小麦Mlo及NBS-LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因Mlo cDNA序列及一个来源于栽培一粒小麦(Triticum monococcum L.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选.结果获得两个表达基因的cDNA克隆.其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%.另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类.白粉菌诱导前后,该片段RT-PCR扩增产物存在差异,表明该片段可能与小麦抗病性相关.利用"中国春"缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上.