A small plasmid for recombination-based screening.

We reported recently the construction of the 4.4-kb R6K-derived pMAD1 plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with ColE1 and therefore permits recombination-based screening of lambda libraries that contain ColE1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the pi-encoding pir gene required for R6K replication. To supply pi [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans, we employed pPR1 delta 22pir116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz, J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger lambda inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1 (1991) 120-123].     

Immunoglobulin complementarity-determining region grafting by recombinant polymerase chain reaction to generate humanised monoclonal antibodies

We describe an approach to rapidly generate humanised monoclonal antibodies by grafting rodent complementarity-determining regions onto human immunoglobulin frameworks using recombinant polymerase chain reaction (PCR) methodology. The approach was applied to grafting a rat complementarily-determining region onto a human framework and amplifying the entire humanised heavy chain. The terminal oligodeoxyribonucleotide primers incorporated restriction sites to allow forced cloning into plasmid vectors for sequencing and expression. No nucleotide errors were introduced into the 1463-bp sequence even after sequential applications of PCR.     

应用反向PCR克隆慢病毒介导的转基因小鼠整合位点序列

目的：为分析慢病毒介导的转基因小鼠中外源基因整合位点的信息,应用反向PCR克隆整合位点序列。方法：小鼠基因组总DNA酶解和自连接后,针对慢病毒载体的特点在LTR附近设计一组特异的PCR引物,优化半巢式PCR的各种参数,提高整合位点序列克隆的效率。结果：克隆了分别携带绿色荧光蛋白（GFP）和转铁蛋白（TF）基因的慢病毒介导的转基因小鼠家系7只小鼠中10个外源基因整合位点序列。结论：本方法可用于慢病毒介导的转基因小鼠整合位点序列的克隆,为分析整合位点与外源基因表达之间的关系等提供了科学依据。     

Amino acid sequence of the N-terminal 139 residues of light chain derived from a homogeneous rabbit antibody

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody (designated BS-1) to type III pneumococci was determined. A combination of methods involving tryptic cleavage restricted to the 2 arginine residues of the molecule and mild acid hydrolysis of a labile peptide bond between the V (variable) and C (constant) regions of the L chain (Fraser et al., 1972) allowed the isolation of two large peptides comprising the entire V region (residues 1-109); these peptides were suitable for automated Edman degradation. The complete sequence analysis of the V region was carried out with only 4mumol of L chain. This material was homogeneous, although minor variant sequences, if present at the 10% value, would not have been detected. The L chain contains 3 intrachain disulphide bridges, whose pairing was established by diagonal electrophoresis: there is one V-region bridge between positions 23 and 88 and one C-region bridge between positions 134 and 194; the third one connects V and C domains between positions 80 and 171. When compared with the basic sequence of human kappa chains, rabbit L chain BS-1 appears to be more similar to the V(KI) prototype sequence than to V(KII) or V(KIII) sequences, where V(KI), V(KII) and V(KIII) represent subgroups I, II and III respectively of V regions of kappa light chains. The V regions of rabbit heavy and light chains are homologous to each other. The presence of two clusters of 3 glycine residues in positions 94-96 and 99-101 respectively is remarkable. Residues 94-96 may be related to antibody complementarity whereas residues 99-101 function probably as a pivot permitting the combining region of the L chain to make optimal contact with the antigenic determinant (Wu & Kabat, 1970).     

Gene conversion of immunoglobulin variable regions in mutagenesis cassettes by replacement PCR mutagenesis.

A technique, Replacement PCR Mutagenesis, was developed to replace one immunoglobulin variable region (V) in a M13 phage cassette with a different, homologous V. This allows the use of the same mutagenesis and subsequent expression vectors for many V regions or V segments. The method combines PCR of V fragments and in vitro mutagenesis. Primers homologous to 3' and 5' ends of both V regions initiate PCR synthesis of the V DNA fragment (donor) that will replace the V region (recipient) in M13. Donor V PCR DNA may originate from mRNA, cloned V genes or genomic templates. The donor V PCR DNA is denatured and annealed to the M13 cassette containing the recipient V to be supplanted. The second strand is synthesized, transfected into bacteria and mutant plaques selected by hybridization. Since restriction sites in primers are not required, altered primer-encoded amino acids are avoided. Further, the PCR donor piece can be of any length if it shares homology with the recipient gene. This allows construction and expression of complete gene replacements and chimeras. This method is also applicable to V "humanization" and studying sets of homologous genes containing polymorphic or evolutionary disparities. The potential uses of the technique are discussed.     

T载体介导的基于attL核心区LR反应的简化快速Gateway克隆系统

Gateway克隆技术已得到广泛的应用。该技术先通过BP反应将目标片段连到带有完整attL特异识别位点的入门载体,然后与终载体通过LR反应得到表达载体。Gateway克隆方法与传统的酶切连接方法相比有快速简单等优点。但是,BP和LR酶都非常昂贵。本研究首先对3个常用Gateway载体的atts特异位点序列比对发现,attL序列核心交换位点“core attL”的21~22 bp长的碱基是保守和必要的。由此,设计含有core-attL序列的引物,通过PCR克隆得到DNA片段并连入pMD18-T载体,然后进行LR反应,可成功得到目标表达载体,并在保守的位点上正确重组。本研究还对其中一个带有绿色荧光蛋白基因的表达载体转化至烟草,能够正常表达该蛋白质。结果表明,通过将含有attL核心位点基因片段连接到pMD18-T载体上,可以省略BP反应而将目标片段连接到终载体上,节约了反应时间和成本。     

Expression vectors for the introduction of highly diverged sequences into the six complementarity-determining regions of an antibody

We prepared three kinds of phagemid vector that permit the simultaneous introduction of highly diverged sequences into six complementarity-determining regions (CDRs) of an antibody (Ab) by the polymerase chain reaction (PCR) with degenerate oligodeoxynucleotide (oligo) primers. The phages expressed either the Fv, single-chain Fv (sc Fv) or Fab form of an Ab fused with a half-molecule of cpIII on the surface of M13 phage. A phage-display library, composed of 2 × 10⁸ independent clones, was constructed; the phages that were specific for hen egg-white lysozyme (HEL) were selected by three rounds of panning; and 20 clones were isolated. The isolated clones consisted of 17 different clones. Among them, 16 clones expressed proteins that were able to bind to HEL. The association constants for binding of the encoded proteins to HEL ranged from 1.48 × 10⁶ to 7.71 × 10⁶/M. These vectors allowed us to prepare many libraries of artificial Ab in which the sequences of six CDRs were very different and reflected the artificial sequences that had been designed for the degenerate oligo that we used as primers for PCR. The libraries should be also useful for the analysis of relationships between the sequences of the CDRs and antigen (Ag) specificity.     

Parallel gene cloning and protein production in multiple expression systems

To quickly find an optimal expression system for recombinant protein production, a set of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously. These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5′‐EcoRI/3′‐XhoI identical in these vectors for ligating with the sticky‐end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax

The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltTBs and gltTBc genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E. coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.     

Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR

To facilitate the rapid cloning and sequencing of rearranged murine heavy-chain variable regions, we have designed a set of universal primers using conserved sequences of leader (signal peptide), framework one and constant regions of the immunoglobulin heavy-chain genes. RNA was extracted from the mouse hybridoma cells secreting monoclonal antibodies: IOR-T3 (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), CB-Fib.1 (anti-human fibrin) and CB-Hep.2 (anti-hepatitis B surface antigen). First-strand cDNA was synthesized and amplified using PCR. The primers successfully amplified correct size fragments from cDNA prepared from all hybridomas. These methods will facilitate the cloning and sequencing of mouse immunoglobulin variable regions.     

Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.     

A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction

A simple and fast method for introducing a series of mutations in cloned DNA has been developed. The polymerase chain reaction (PCR) has been used for site-directed mutagenesis. Because mutations can be introduced only within the primer sequences used for PCR, a suitable restriction site in the vicinity of the mutated nucleotide is required to permit recloning. Several methods have been devised to overcome this limitation. Our present method is a modification of the overlap extension method [Ho et al., Gene 77 (1989) 51-57], and has some advantages over this and other published methods. In our method, three common primers and a series of primers specific for various mutations are chemically synthesized. Once the proper oligodeoxyribonucleotides are selected as common primers, each mutation requires only one additional primer. Therefore, this method is very useful for introducing many mutations in various sites of the target DNA. We describe our protocol for the site-directed mutagenesis and an example of the introduction of several mutations in the hen egg-white lysozyme-encoding gene.     

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3 months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.     

小鼠4.5S RNA逆转座子对Luc基因表达的影响

利用ＲＴ－ＰＣＲ方法,从小鼠肝脏组织总ＲＮＡ中扩增出４．５ＳＲＮＡ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,酶切鉴定并测序。然后将该序列插入以Ｌｕｃ基因作为报道基因的表达载体ｐＳＶｌｕｃ２０的ＰｖｕⅡ位点,构建了含４．２ＳＲＮＡ逆转座子的表达载体ｐＳＶｌｕｃ２０－４．５Ｓ。脂质转染法将表达载体导入小鼠骨髓瘤细胞ＮＳ－１、ＳＰ２／０和人乳腺癌细胞Ｂｃａ６１。结果表明,小鼠４．５ＳＲＮ     

基于PIPE现象的质粒克隆位点序列设计与功能评价

分子克隆是现代生物学研究的核心技术之一,是基因工程、蛋白质工程中的重要手段。为提高分子克隆实验的操作效率,本研究设计并合成基于聚合酶引物不完全延伸（polymerase incomplete primer extension,PIPE）现象的质粒克隆位点序列。并以此为基础统一相关引物的设计方案,避免传统酶切--连接法中需针对不同载体MCS序列设计不同引物的缺点。该方案利用13 bp定长接头序列,在同一体系中使用2对引物、2种线性化模板同时扩增载体和插入片段,通过20个循环,在1次PCR过程中即合成可供转化使用的带缺口质粒产物。在NEB Q5酶系统中,利用此法将3种荧光素酶序列插入pET-15b及pET-21b(+)载体,均获得成功。且利用商品化感受态细胞（转化效率 > 5×108 cfu/μg）转化后所获得转化子数量均在300个以上,其中含插入片段的阳性克隆比例可达85%以上。基于本方案的设计及作用原理,可将其应用于10 kb以内载体和插入片段的快速重组。且具有通用性强、耗时少、阳性克隆得率高和成本低等优点,是传统DNA重组方法的有益补充,可作为各实验室的常规分子克隆手段之一。     

Domain libraries: Synthetic diversity for de novo design of antibody V-regions

A completely synthetic gene library encoding the variable light (VL) immunoglobulin domains has been constructed in vitro. The library was constructed by assembling a set of six oligodeoxyribonucleotides (oligos) using the polymerase chain reaction (PCR). Three out of the six overlapping oligonucleotides were synthesized with randomized complementarity determining regions (CDR) with the codon pattern, (NNS)_n, where N is any of the four nucleotides (nt) and n is the number of codons with variation in the CDR. The framework regions, taken from the D1.3 anti-lysozyme antibody (Ab), were kept intact. Overlapping regions of approx. 20 nt, together with two additional flanking primers carrying the desired restriction sites, allowed the construction of a library in one single PCR reaction. The VL library was cloned into the phage display vector pEXmide3, and ten randomly picked clones were sequenced. These sequences exhibited complete diversity in all the three CDR and the codons for five canonical amino acid (aa) residues were kept intact and identified. Seven clones contained the full-length gene for the VL domain while deletions were observed in three clones. The restricted use of nt at the third position successfully avoided the stop codons TGA and TAA, whereas the stop codon TAG is read as Gln in an amber suppressor strain. We call this synthetic Ab diversity Domain Library, and it represents an example of syntheticlibraries with extensive, multiple randomized sequences. The use of Domain Libraries opens up the possibility for design in Ab engineering, e.g., additional CDR regions can be added or their length varied. Furthermore, the use of synthetic gene libraries, constructed with the Domain Library strategy, is not limited to the construction of synthetic Ab fragments, but can be used in the design of other types of proteins.     

Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha, beta, and gamma subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.