用RACE结合cDNA文库筛选的方法获取新的锌指蛋白基因

大多数有重要功能的蛋白质都含相应的由保守氨基酸顺序组成的功能结构域。本文首先根据蛋白质功能结构域保守氨基酸序列设计简并引物,用PCR方法扩增出基因EST序列,再利用改进的快速扩增cDNA末端（RACE）方法从cDNA文库中扩增出基因非同源部位,然后以非同源序列为探针,筛选cDNA文库。利用此方法成功地从人骨髓cDNA文库中克隆到几个编码锌指蛋白并代表原有EST的新的全长cDNA。这一策略也应适用于筛选编码具有其他序列保守性功能结构域蛋白的基因。 Abstract:Most of the important functionally proteins contain the corresponding function domains that consist of conserved amino acid sequences.The study provided a method to identify novel genes that encode proteins containing important functionally domains with conserved sequences.First,primers were designed according to the sequence of the cDNA library vector and the ESTs that have been obtained by reverse PCR and degenerate primers encoding Zinc finger domain.The cDNA library DNA was used as template for PCR amplification.The amplified fragment that contains nonhomologous sequences of the cDNA was inserted into pGEM-T easy vector.The fragment was recovered and used as a probe for screening the cDNA library.Several cDNAs with full length that encode proteins with Zinc finger domain and represent the original ESTs have been successfully cloned from a human bone marrow cDNA library.This strategy can also be used in screening genes that encode proteins containing differential function domains with conserved sequences.     

野生与笼养绿孔雀种群的随机扩增多态DNA研究

利用随机扩增多态DNA（RAPD）技术对野生14只和笼养18只绿孔雀（Pavo muticus）个体进行了种群遗传多样性分析。用23个随机引物,野生与笼养绿孔雀分别获得161和166个扩增片段,计算发现野生与笼养绿孔雀的种群内平均相对遗传距离分别是0.0555和0.1355,两种群间的为0.1635;两种群的Shannon多样性指数平均分别是0.4348和1.0163,有显著性差异。以上分析都显示野生绿孔雀的遗传多样性很低。用UPGMA法聚类显示两个种群都是分别来源于两个家系,可据此进行繁育管理。 Abstract:Random-amplified polymorphic DNA(RAPD) was used to investigate the genetic diversity of the population of 14 wild green peafowl and 18 captive green peafowl(pavo muticus).Total of 161 and 166 bands were obtained respectively,and 23 random primers were used to amplify the genomic DNA of the wild and captive green peafowls.The average relative hereditary distance of the wild and captive green peafowls is 0.0555 and 0.1355 respectively;and the Shannon diversity index is 0.4348 and 1.0163 respectively.There is a prominent differentia between the two populations by T-Test of HO.All the analyses above show that the genetic diversity is very low in wild green peafowl.It tells us that the two populations come from two families by using UPGMA,which can be useful in the breeding management in the future.     

云南汉族群体FIBRA、DHFRP2、ACTBP2基因座多态性及其法医学应用

采用Amp-FLP分型方法,调查云南汉族群体FIBRA、DHFRP2、ACTBP2基因座的遗传多态性,并将其应用于法医学实践。200份EDTA抗凝血采自昆明地区无血缘关系汉族个体,采用酚—氯仿法提取DNA;法医物证实际检案及亲子鉴定检材取自昆明医学院法医系物证教研室检案,各种动物血痕取自动物中心,采用酚—氯仿法或Chelex法提取DNA,PCR扩增,非变 性聚丙烯酰胺凝胶垂直板电泳,硝酸银染色分型。结果表明,FIBRA、DHFRP2、ACTBP2基因座分别观察到15、7、13个等位基因,基因型数分别是57、25、61。3个STR基因座的杂合度（H）分别为：0.8940、0.8174、0.9130;多态信息容量（PIC）分别是:0.8908、0.8045、0.9117;个人识别力（Dp）分别是：0.9733、0.9416、0.9772;非父排除率（Epp）分别是：0.7994、0.6542、0.8348,基因型频率分布均符合Hardy-Weinberg平衡。20个家系调查结果表明,3个基因组均符合孟德尔遗传规律。 Genetic Polymorphism of FIBRA,DHFRP2 and ACTBP2 and Their Forensic Application in Yunnan Han Population JING Qiang,NIE Sheng-jie Department of Forensic Medicine,Kunming Medical College,Yunnan Province 650031,China Abstract:To investigate the genetic polymorphism of FIBRA,DHFRP2 and ACTBP2 in Yunnan Han population as well as their application in forensic science,EDTA-blood specimens were collected from 200 healthy individuals.The DNA were extracted either by the Chloro form,phenol method or by the Chelex-100 method.The PCR products were analyzed by PAG vertical electrophoresis,following by silver staining.All gene frequencies,discrimination power (DP),exclusion of paternity probability (EPP),heterozygosity (H),polymorphisms information content (PIC),matching probability (PM) as well as the Hardy-Weinberg test were calculated.The obtained data are beneficial in the understanding of population genetics of the three STR loci in Yunnan Han population and the results suggest that these loci are valuable genetic markers for paternity testing and personal identification in forensic science practice. Key words：short tandem repeat; Amp-FLP; genetic polymorphism     

PCR反应混合液的冻干处理及其应用

本研究将除模板DNA以外的PCR反应混合液,经过真空浓缩冻成干粉状后,分别置于室温和4℃贮存,经过一定的贮藏时间后进行PCR反应,发现经冻干处理的样品能在较长的贮存时间内保持扩增活性,这将给实际工作带来很大的便利。 Abstract:In this paper we introduce a new storage method of PCR ingredient.PCR mixture except DNA template has been frozen to dry powder by the DNA-Plus system.This kind of powder was stored at room temperature or 4℃.PCR has been run in different period of storage.It was discovered that the samples of lyophilization could keep activity for a long time.     

大肠杆菌FC40系统静止期突变中的F因子转移

本研究采用大肠杆菌GM133 rifr细胞和营养收集细胞HB214 strr进行适应性突变实验。在混合30min和2d 后添加链霉素杀死GM133基因型细胞,继续培养5d后,在选择平板上出现了一定数量的lac+strr基因型回复突变菌落。根据这些突变菌落的数量,估计在lac+突变产生之前,GM133和HB214细胞之间的接合频率分别为0.07%和7.47%。在培养了7d的选择平板上添加含链霉素的M9选择培养基,2d 后也观察到大量发生lac+突变但没有形成肉眼可见菌落的营养收集细胞。此外,在lac+突变发生后,也有F因子从GM133细胞转移进入HB214细胞。这些事实表明,在FC40系统的适应性突变实验中发生了真正的F因子转移。 Abstract:The experiment of adaptive mutation was performed by using Escherichia coli GM133 rifr as test cells and HB214 strr as scavenger cells.Transfer frequency between GM133 and HB214 was estimated,based on the number of revertants appeared on the selective plates when GM133 were killed by addition of M9 selective medium containing 100μg/mL of streptomycin at different time.After 30 minutes the cells of GM133 and HB214 were mixed,the estimated transfer frequency was about 0.07%,and two days,7.47%.After selection of 7 days,some HB214 cells with F` factor from GM133 cells and lac+ mutation were observed,but these cells failed to form the colonies which can be seen by the naked-eye.It was demonstrated that actual F` factor transfer events from test cells GM133 to scavenger cells HB214 occurred during the selection.     

用整合微流控芯片系统分析黄山松RAPD片段多态性

随机扩增多态DNA（randomly amplified polymorphic DNA,RAPD）标记可快速提供连锁信息,特别是在针叶树单倍体的大配子体中RAPD基因型也能得以确定［3］。在对多态性的RAPD片段进行分析时,传统的平板凝胶电泳分析法因人工操作,其有效的鉴别受到一定程度的限制,只能得到一些有关RAPD片段半定量信息。我们将整合微流控芯片系统作为一种新的工具运用于黄山松的多态性RAPD片段分析中。发现基于芯片的检测方法比琼脂糖凝胶电泳方法灵敏度更高,所需的样品量要少得多,所需的时间仅为其1/4。并且能自动对DNA片段进行定性、定量分析。它是一种高效、灵敏、迅速、重复性好的检测新技术。 Abstract:Randomly amplified polymorphic DNA (RAPD) markers quickly provide linkage information［1~2］,especially in conifers where haploid megagametophytes can be used for genotyping［3］.Traditionally use of slab gel electrophresis results in qualitative data that can be manually manipulated to gain semiquantitative information about the polymorphic RAPD fragments.We have proposed the use of an integrated microfluidic chip-based system as a new tool in the analysis of polymorphic RAPD fragments.The chip-based method was found to be very sensitive,requiring much less sample and only quarter the time compared to the agarose gel method.The automated data analysis sizes and quantitates the DNA fragments,thus yielding a more thorough,reproducible,sensitive,and rapid analysis.     

4个Y-STR基因座银染复合扩增

建立一个复合扩增体系,同时扩增DYS391、GATA-A4、GATA-A10和GATA-H4 4个Y染色体特异性的STR基因座,并用聚丙烯酰胺凝胶电泳和银染显色技术进行基因分型。采用该复合扩增系统调查广东汉族311名无关男性个体的单倍型频率,这4个基因座分别检出5、7、6和5个等位基因,其基因多样性分别为0.4623、0.6972、0.7173、0.6015。共检出98种单倍型,单倍型基因多样性为0.9755。该复合扩增体系在建立Y染色体STR数据库,研究群体遗传和进行法医学鉴定有重要意义。 Abstract:A multiplex PCR system has been developed to amplify 4 Y-chromosome specific short tandem repeats(STR),DYS391,GATA-A4,GATA-A10 and GATA-H4,simultaneously.PCR products were separated by polyacrylamide gels electrophoresis followed by silver stain.When 311 unrelated males from the Han population in Guangdong were detected by the multiplex system,DYS391,GATA-A4,GATA-A10 and GATA-H4 showed 5,7,6 and 5 alleles respectively.Total 98 haplotypes could be identified.Gene diversity value for the 4 STR was 0.4623,0.6972,0.7173 and 0.6015 respectively.The gene diversity value for the haplotypes of the 4 Y-STR reached 0.9755.The four Y-STR multiplex system will be very powerful for establishing Y-STR database,exploring human origin,paternity testing and personal identification.     

巨穗小麦种质中外源遗传物质的细胞遗传学和分子生物学鉴定

利用C分带、基因组原位杂交并结合分子生物学手段,对12份巨穗小麦种质材料中的外源遗传物质进行了检测.结果表明,12份材料染色体数均为42,其中5份材料均具有一对小麦-黑麦(Triticum aestivum-Secalecereal)1BL/1RS易位染色体和一对中间偃麦草(Agropyron intermedium Garten)染色体、3份材料只具有一对中间偃麦草染色体、3份材料只具一对1BL/1RS染色体、1份材料无1BL/1RS和中间偃麦草染色体.进一步细胞学分析表明,此中间偃麦草染色体代换了普通小麦(Triticum aestivum L.)中的2D染色体,因其良好的同源补偿性,表示为2Ai.同时对2Ai在巨穗小麦种质中存在的遗传学意义及小麦遗传改良中的应用进行了讨论.     

猪繁殖与呼吸综合征病毒N基因的克隆及高效表达*

本试验参照GenBank公布的Porcine reproductive and respiratory syndrome virus(PRRSV)VR2332株的核苷酸序列,设计并合成了一对引物,应用RT-PCR方法扩增出了PRRSV的核衣壳蛋白基因(N基因).在对N基因及pET32a载体双酶切后进行连接,构建了高效原核表达载体pETN.将pETN重组质粒转化BL21(DE3)宿主菌后,对培养条件及诱导表达条件(IPTG最佳浓度、作用时间)等影响表达的因素进行优化,实现了PRRSV核衣壳蛋白基因的高效表达.     

滚环DNA扩增的原理、应用和展望

滚环DNA扩增 (rollingcircleDNAamplification ,RCA)是一种等温信号扩增方法 ,其线性扩增倍数为 1 0 ⁵,指数化扩增能力大于 10⁹,产生的扩增产物连接在固相支持物 (如玻片、微孔板等 )表面的DNA引物或抗体上。RCA是一种适合在芯片上 (on chip)进行信号扩增的新技术 ,它既能提供研究分析的敏感性和特异性 ,又能保持立体分析的多元性。RCA亦是一种痕量的分子检测方法 ,可用于极其微量的生物大分子和生物标志的检测与研究