The development of a novel vector for construction of a full-length cDNA library

A new original vector pEM-(dT)₄₀(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The vector pGEM-(dT)₄₀f(+) is initially transformed into single stranded and then into a linear form and its (dT)₄₀ tail at the 3′-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector circularization and synthesis of the second strand cDNA. This approach has the following advantages: (1) it significantly simplifies cDNA library construction, which includes three steps; (2) full-length cDNA library construction is achieved by adding a (dC)_n homopolymer tail to the 5′end; (3) preparation of a clone library requires a few milligrams of total RNA; (4) it is possible to obtain cDNA clones up to 10 kbp; (5) it does not require PCR reaction (which can induce artifact mutations in cDNA sequences); (6) this approach does not employ restrictase treatment and chimeric cDNA products are not formed.     

Solid-phase cDNA library construction, a versatile approach.

A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for most purposes.     

Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.     

“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     

Four-way ligation for construction of a mammalian cell-based full-length antibody display library

A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.     

FULL-malaria: a database for a full-length enriched cDNA library from human malaria parasite, Plasmodium falciparum

FULL-malaria is a database for a full-length-enriched cDNA library from the human malaria parasite Plasmodium falciparum (http://133.11. 149.55/). Because of its medical importance, this organism is the first target for genome sequencing of a eukaryotic pathogen; the sequences of two of its 14 chromosomes have already been determined. However, for the full exploitation of this rapidly accumulating information, correct identification of the genes and study of their expression are essential. Using the oligo-capping method, we have produced a full-length-enriched cDNA library from erythrocytic stage parasites and performed one-pass reading. The database consists of nucleotide sequences of 2490 random clones that include 390 (16%) known malaria genes according to BLASTN analysis of the nr-nt database in GenBank; these represent 98 genes, and the clones for 48 of these genes contain the complete protein-coding sequence (49%). On the other hand, comparisons with the complete chromosome 2 sequence revealed that 35 of 210 predicted genes are expressed, and in addition led to detection of three new gene candidates that were not previously known. In total, 19 of these 38 clones (50%) were full-length. From these observations, it is expected that the database contains approximately 1000 genes, including 500 full-length clones. It should be an invaluable resource for the development of vaccines and novel drugs.     

米曲霉(Aspergillus oryzae) RIB40全长cDNA文库的构建

【目的】构建米曲霉RIB40的全长cDNA表达文库,为米曲霉功能基因的开发以及次生代谢产物合成途径相关基因的筛选与克隆奠定基础。【方法】采用RNAiso法从米曲霉RIB40菌体中提取总RNA。选用PolyATract mRNA Isolation System Ⅲ试剂盒分离纯化mRNA。以5μg mRNA为模板,按照ZAP-cDNA Synthesis Kit试剂盒说明书要求合成单、双链cDNA,使用CHROMA SPIN-400柱离心层析纯化后连接于Uni-ZAP XR表达载体上,体外包装后转染Escherichia coli XL1-Blue宿主菌。【结果】构建了米曲霉RIB40的全长cDNA文库,初级文库滴度约为2.96×106 CFU/mL,重组率约为97.8%,插入片段平均长度大于1.5 kb,达到一个高质量cDNA文库的要求。文库扩增后,滴度达到3.4×1010 CFU/mL。【结论】米曲霉RIB40全长cDNA表达文库的成功构建,将会对米曲霉基础生物学研究及相关基因的筛选与克隆奠定基础。     

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.     

SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

Background  

cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library.