A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation

A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail.     

Directional cloning of cDNA using a selectable SfiI cassette

To increase the efficiency of directionally cloning cDNA, we have constructed a pair of vectors and devised a cDNA cloning strategy that improves upon previously published methods. The vectors, pLIB: AZ and pLIB: ZA, have two unique (distinct religation specificities; GGCCN/NNNNGGCC) SfiI sites (SfiI.A and SfiI.B) flanking a stuffer fragment which contains the tetracycline-resistance element. These vectors permit the directional cloning of cDNA in both sense (pLIB: AZ) and antisense (pLIB: ZA) orientations relative to the promoter for phage T3 RNA polymerase. cDNA that was synthesized using a primer with a 5' sequence of a SfiI.B site followed by an oligo(dT)16 3' tail was then ligated to an adaptor with the sequence of a SfiI.A site produced directional molecules that could be cloned into the pLIB vectors. Complex libraries with 10(7) members were produced from as few as 6 x 10(5) cells. The SfiI sites and stuffer can be subcloned as a cassette to permit directional cloning in other vectors, as there are several restriction enzyme sites flanking this region to the 5' and 3'.     

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.     

Direct cloning of cDNA inserts from lambda gt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage lambda gt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for lambda gt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from lambda gt11 phage were cloned directly into the pBR322 plasmid vector, bypassing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage lambda vectors when bacteria containing endogenous pBR322 are used as host cells.     

Direct cloning of cDNA inserts from λgt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage λgt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for λgt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from λgt11 phage were cloned directly into the pBR322 plasmid vector, by-passing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage λ vectors when bacteria containing endogenous pBR322 are used as host cells.     

Use of matrix-immobilised recombinant plasmids to purify chain-specific rabbit globin complementary DNAs.

The cloning of DNA sequences in plasmid recombinants has made it possible to amplify specific sequences to an extent that they can be used for preparative purposes. We describe the use of rabbit globin DNA sequences cloned in the plasmid pCR1 and covalently bound to Sepharose 4B for the purification of chain-specific rabbit alpha- and beta-globin cDNAs. These purified probes were then used to estimate the length of the alpha- and beta-globin DNA sequences inserted into the recombinant plasmid. The technique should allow the rapid isolation of sequence-specific cDNA, RNA and genomic DNA.     

Cloning of randomly sheared DNA fragments from a phi 105 lysogen of Bacillus subtilis identification of prophage-containing clones

A gene bank for Bacillus subtilis has been developed by cloning randomly sheared DNA fragments of a B. subtilis (phi 105) lysogen DNA in Escherichia coli employing the pMB9 plasmid vector. The DNA was inserted by the oligo(dA)-oligo(dT) method, and the average insert size of the cloned DNA was 7 kilobase pairs (kb). Three clones have been identified which carry DNA from the phi 105 prophage. None of these clones contain the phage-chromosome junction.     

利用SMART方法快速克隆异黄酮代谢途径多基因的研究

采用改进的酸酚法提取高质量的大豆叶片RNA,利用SMART思想和方法构建大豆叶片全长cDNA文库,直接以一级库液稀释液为模版进行PCR,快速克隆得到异黄酮代谢途径相关的5个基因。与传统的从DNA、RNA出发克隆基因,以及构建文库再进行基因筛选的克隆方法相比,该方法得到的基因均为全长基因,适用于快速、简便的进行多基因全长克隆。     

Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA.

A simple strategy is described for the generation of catalytic hammerhead-type ribozymes (Rz) that can be used as highly specific endoribonucleases to cleave a particular target RNA. The technique requires that a cloned cDNA fragment is available which encodes at least a part of the target RNA. About 25 different restriction recognition sequences can be utilized to incorporate specifically designed DNA cassettes into the cDNA. Besides some nucleotides which are specific for a certain restriction site, the DNA cassettes contain a sequence corresponding to the catalytic domain of the hammerhead Rz and, optionally, selectable marker genes, that are removable. The resulting recombinant DNA constructs permit the in vitro and in vivo synthesis of novel 'catalytic antisense RNAs' or 'antisense Rz (Az)', which combine two features: (i) they bind like antisense RNA to their specific substrate RNA, and (ii) they cleave their target as hammerhead Rz do. The utility of the strategy to generate Rz was demonstrated experimentally by incorporating a synthetic SalI-specific DNA ribozyme (Rz) cassette into a unique SalI site of a cloned cDNA fragment of plum pox virus (PPV), which is a single-stranded positive sense plant RNA virus, belonging to the group of potyviruses. The resulting Az constructs delivered Az that were directed against the PPV (+) or (-) RNA, respectively, which cleaved their corresponding target RNAs in the expected manner. Besides the synthetic Rz cassette, a comparable SalI-specific Rz cassette, that had been prepared from a specifically designed plasmid and that contained the tet gene inserted into the sequence of the catalytic domain of the Rz, was also incorporated into the SalI site of the PPV cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of an equalized cDNA library from Arabidopsis thaliana

Using a kinetic approach, a cDNA library composed of almost equal representations of all genes expressed in the aerial parts of 2-week-old Arabidopsis was constructed. A cDNA was synthesized with an oligo dT primer containing a Not l site. A linker containing the nucleotide sequence of Sse 8387I which recognizes octanucleotides was added at the ends of the synthesized cDNA. The cDNA was amplified by the polymerase chain reaction (PCR), denatured, and reassociated under modified conditions. Thereafter, the remaining single-stranded DNA was converted to double-stranded DNA and amplified by PCR. These equalization steps were repeated three times and the products were cloned unidirectionally into a plasmid vector. Equalization was evaluated by colony hybridization and DNA sequencing. This approach will be applicable to construct a cDNA library suitable for subtraction, differential screening, and expression screening, especially for mRNA species present at very low concentrations in a few cells of a specific tissue.     

Sequences hybridizing to mRNA, oligo(dT) and dsRNA from pre-mRNA are contiguous in the cloned mouse DNA fragments.

Fragments from the DNA of mouse embryos produced by restriction endonucleases HindIII were cloned in pBR322 plasmid and examined for the ability to hybridize in situ with [32P] labeled cDNA synthesized from the polysomal poly(A)+mRNA template. Several of the selected clones were examined for the presence of specific sequences inside the cloned mouse DNA fragments by the blotting procedure of southern [1]. The data obtained indicate that the majority of the cloned mouse DNA fragments contained sequences hybridizing with cDNA, oligo(dT) and double-stranded regions from pre-mRNA. The results of hybridization experiments and double digestion with HindIII+HaeIII endonucleases provide evidence that these sequences could be contiguous in the given restriction DNA fragments.     

应用逆转录聚合酶链反应法扩增类胰岛素生长因子Ⅱ基因

利用酸性异硫氰酸胍-酚-氯仿一步法从人胚胎组织中提取总RNA,经Oligo(dT)纤维柱分离纯化出mRNA。用逆转录与聚合酶链反应相结合的RT-PCR法,扩增出人类胰岛素生长因子Ⅱ(IGFⅡ)的cDNA片段,在限制性内切酶Sma Ⅰ存在的连接体系中,将扩增出的cDNA片段克隆进PUC12的Sma Ⅰ位点处。经限制性内切酶EcoR Ⅰ、Sal Ⅰ、Eco47Ⅲ酶切鉴定其方向。以重组质粒的双链DNA为模板,用末端终止法测定其全部核苷酸顺序,证实其核苷酸编码的IGFⅡ在氨基酸顺序上与文献报道的相同。     

Expression cloning of a sheep adreno-ferredoxin using the polymerase chain reaction.

In addition to the selective amplification of cDNA from total RNA by the PCR method, the distinctive properties of ferredoxin-expressing colonies can be used for cloning a ferredoxin cDNA. This strategy for cloning and expressing cDNA in E. coli was applied to a sheep adreno-ferredoxin. The expressed sheep ferredoxin showed a spectral pattern typical of [2Fe-2S] proteins. The amino acid sequence deduced from the DNA sequence showed that the mature form of sheep ferredoxin consists of 128 amino acid residues. This rapid and simple method for cloning and expressing cDNA can be applied to other ferredoxins.     

人钠/碘同向转运体基因cDNA的克隆与序列测定

为研究人钠／碘同向转运体(hNIS)的生物学性能和用于肿瘤放射性碘治疗的可能性,运用反转录聚合酶链反应(RT—PCR)从人甲状腺组织总RNA中扩增出hNIS基因cDNA序列,将其克隆至pUCm-T载体中。序列分析证实克隆片段与献报道的hNIS基因cDNA序列完全一致,说明已成功克隆到hNIS基因cDNA。