Clustering cDNA sequences

A set of programs has been written to quantify the similaritiesbetween large numbers of cDNA sequences. This information isused to cluster similar sequences together. The main programcan cluster thousands ofcDNA sequences per day using a novel,computationally inexpensive algorithm. The clustering informationis kept in a small index file so that disk storage requirementsare negligible. Using this index file, subsidiary programs createvarious views and statistical summaries of the entire cDNA sequencecollection.     

“电子”cDNA文库筛选指导基因的全长cDNA克隆

“电子”ｃＤＮＡ库筛选主要是指通过采用生物信息学的方法延伸表达序列标签（ＥＳＴ）序列,以获得基因的部分及至全长ｃＤＮＡ序列,避免或部分避免构建与筛选ｃＤＮＡ库等烦琐的实验室工作。该方法具体体现了ＥＳＴ数据库的迅速扩张已导致识别与克隆新基因的策略发生革命性的变化。ＥＳＴ序列ＺＡ７３为本实验室克隆到的可能参与辐射致气管上皮细胞恶性转化过程的基因片段,本研究采用“电子”ｃＤＮＡ库筛选的方法对其可能     

Construction and Screening of Chickpea cDNA Library with Heterologous Legumin cDNA Probe

A cDNA library was constructed from developing chickpea cotyledons in the expression vector λ gt 11. The library when plated on X-gal and IPTG showed about 35% recombinants. These recombinants were screened using pea legumin cDNA probe. Of a total of 4 × 10⁴ cDNA clones screened, 20 clones showed homology to legumin probe. The presence of cDNA insert in these clones was further confirmed by Southern blot hybridization.     

大鼠肾脏组织cDNA文库的制备和葡萄糖转运蛋白cDNA克隆的筛选

 大鼠的肾脏组织经匀浆后,提取纯化总RNA和mRNA合成cDNA,进行甲基化,加人工接头,最后连入载体(λgt11)和外壳蛋白包装步骤制成肾脏组织的cDNA文库。插入载体的cDNA长度为0.5kb到8kb之间。经反复稀释,测定出该文库的效价为1.5×10~7pfu/mL。     

Cloning of PCR-amplified total cDNA: Construction of a mouse oocyte cDNA library

We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)⁺ RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)⁺ RNA in this mammalian oocyte.     

Removal of known, abundant cDNA species by specific double-stranded cDNA synthesis-based subtraction

An efficient and simple method for removing known, abundant cDNA species from the cDNA pool of highly differentiated cells is reported. The method involves preparation of sscDNA, followed by dscDNA-synthesis of known, abundant cDNA species led by specific primers and removal of the synthesized dscDNA with hydroxyapatite. By using this method, the globin cDNAs were reduced to less than 10⁻⁵ of their original abundance. The results suggest that this method may facilitate the isolation of new genes from specific cells or tissues.     

Structure of mouse interferon-beta cDNA clones: sequence rearrangements in a cloned cDNA

A fraction enriched in interferon (IFN) mRNA was prepared from mouse C243-3 induced cells and was used for the construction of a cDNA library. Two plasmids were obtained after screening by differential colony hybridization and IFN mRNA hybridization-selection and translation. The nucleotide sequences of the cDNA inserts revealed that both were partial copies of IFN-beta mRNA. The cDNA 861 corresponds to the entire 3' nontranslated region of the mRNA while the cDNA 2939 consists of rearranged translated regions of IFN mRNA. A mechanism for the rearrangement events during cDNA synthesis is proposed. A chromosomal DNA fragment hybridizing to cDNA 2939 was identified by screening a mouse genomic library.     

一种新的cDNA末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物。利用一种新的cDNA末端快速扩增方法（SMART RACE）扩增该EST的5′末端,并进行克隆测序,与cDNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA。结果表明：SMAR RACE是一种简便、有效的克隆cDNA5′末端未知序列的技术。     

Construction of Rice cDNA Library and Partial Sequence Analysis of 100 Randomly Selected cDNA

A cDNA library of rice (Oryza sativa ssp. indica cv. "Guangluai 4") etiolated shoot was constructed using Lambda ZAP Ⅱ vector. After analysing the partial sequences of 100 randomly selected clones and database comparison to rice and other plants, 13 % cDNA clones could be identified and 12 % cDNAs had high degree of sequence similarity to partial sequence from rice or other species, whose function is still unknown. The remaining 75% cDNAs showed little or no similarity to genes in the database and might represent novel genes. It demonstrates the suitability of this library for large-scale sequencing from which more information of functional genes will result.     

快速老化小鼠海马差异表达cDNA芯片的制作

老年性痴呆(Alzheimer’s disease,AD)是老年人群中最普遍的痴呆类型,是一种神经退行性紊乱疾病,目前临床上还没有有效的治疗方法。快速老化小鼠亚系P8(senescence-accelerated mouse prone8,SAMP8)是研究增龄相关性认知缺陷机制以及研究脑老化机制的良好动物,同时也是研究AD较为理想的实验动物模型之一。cDNA芯片技术可以同时规模研究成千上万个基因的表达,尤其适于AD这种多机制、多靶标、多途径的复杂疾病的研究,为了揭示AD的发病机制,发现用于治疗AD的药物靶标,以SAMP8和SAMR1海马抑制消减cDNA文库中的cDNA片段为材料,以β-actin和G3PDH为内参,设计了16×(1×14)点阵方案,并点制了含有3136个点的SAM海马差异表达cDNA芯片。芯片背景均匀一致,点的大小均一,排列规则整齐。在靶分子与探针杂交过程中,进行了杂交条件和洗涤芯片的优化。将杂交结果进行统计分析,选择差异表达的cDNA进行测序并进行生物信息学分析,用实时定量RT-PCR对部分基因的表达进行了验证,检测了芯片筛选结果的可靠性。该芯片的成功制备为进一步进行差异表达基因的筛选和研究提供了良好的手段,并将成为揭示SAMP8脑老化和AD发病机制的有力手段。     

Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5'-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5'-end non-coding region and a poly(A) signal AATAAA at the 3'-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3' region after poly(A) signal.     

Cloning of PCR-amplified total cDNA: construction of a mouse oocyte cDNA library.

We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)+ RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)+ RNA in this mammalian oocyte.     

假密环菌cDNA文库的构建及其阿拉伯糖苷酶基因的克隆(英文)

用SMART技术构建了载体为λTriplEx2的假密环菌的表达型cDNA文库。文库滴度为1．0×106pfu／ml,重组率约为98．3％,扩增后滴度为3．1×108pfu／ml,容量约为4．2×1010。从文库中随机挑选176个克隆进行5’端测序,得到147条表达序列标签(EST),并将测序结果提交到EMBL数据库。随机测序结果表明:插入片段长度均在200-800之间。测序结果经过生物信息学分析,发现有43条序列与已知序列有明显同源性,其中序列AJ620046与多形拟杆菌的阿拉伯糖苷酶序列有很高的序列一致性。用SMART-RACE技术成功获得了AJ620046的全长cDNA,克隆了AJ620046的开放阅读框AF,并成功构建了重组质粒pHIL-S1-AF,在毕赤酵母菌中进行了初步表达。     

一种新的cDNA 末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物,利用一种新的cDNA末端快速扩增方法(SMARTRACE)扩增该EST的5′末端,并进行克隆测序,与mRNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA.结果表明,SMARTRACE是一种简便、有效的克隆cDNA5′末端未知序列的技术. Abstract:To clone the full-length cDNAs of genes related to spermatogenesis,ESTs obtained by mRNA differential display were used to design gene-specific primer.Then SMART RACE was performed to obtain the 5′ region of these ESTs.After cloning,sequencing and splicing with ESTs obtained by mRNA differential display,three full-length cDNAs were obtained.The results indicate that SMART RACE is a simple and an effective technique for cloning 5′-end unknown sequence of gene.     

低密度cDNA芯片技术的优化

为了建立稳定的低密度cDNA芯片技术平台,研究靶基因的最适长度、浓度、点样溶液种类及杂交反应动力学,并了解该芯片的重复性与可靠性.结果表明,杂交具有较好的特异性,不同长度(189～1078bp)、浓度(0.5g/L、1.0g/L、1.5g/L)的同一靶基因杂交信号强度无明显差别;以50%DMSO为点样溶液者杂交信号最好(P=0.0001).60℃杂交18h信号最佳(P<0.001).重复2次检测结果差异无显著性(P=0.348),重复性较好,其相关系数为0.588.与RT-PCR结果相比,相关系数为-0.778(P<0.0001),特异性为100%,灵敏度为80%(16/20),可靠性较好.