Construction of linear functional expression elements with DNA and RNA hybrid primers: a flexible and fast method for proteomics

Two methods of constructing linear functional expression elements (LFEE) using hybrid DNA and RNA primers in DNA amplification for rapid gene expression are described. In both methods, it is not necessary to have additional transformation or bacterial propagation. The promoter, open reading frame (ORF) and terminator are amplified using Pfu or Taq DNA polymerase. Three elements containing DNA or RNA overhang are covalently ligated by T4 DNA ligase. The recombinant molecule is amplified with element-specific primers. The LFEE can be generated by both methods in a few hours and can be expressed in mammalian cells.     

一种高效构建同源重组DNA片段的方法——融合PCR

融合PCR技术（fusion PCR）采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以三个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行三个片段及四个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。     

Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase, N.Bpu10 I, and exonuclease III

A method to assemble linear expression elements for rapid gene expression is described. Primers containing target specific sequences and N.Bpu10 I nickase recognition sites were used to amplify promoter, open reading frame and terminator fragments. Amplified fragments were treated with N.Bpu10 I nickase and exonuclease III to generate overhangs for directional ligation. These fragments were ligated and further amplified with element-specific primers. The amplified DNA was transfected into mammalian cells for gene expression.     

Thermostable DNA polymerase from Thermus thermophilus B35: cloning, sequence analysis, and gene expression

The nucleotide sequences of three thermostable DNA polymerase (Taq, Tth, and Tfl) genes were analyzed and high conserved regions typical for this polymerase family were identified. Using primers for one of the conserved regions, the genomic DNA fragment of T. thermophilus B35 strain was amplified. The resulting fragment was cloned into a plasmid and used as a hybridization probe with digests of T. thermophilus B35 DNA cleaved by different restriction endonucleases. A restriction DNA fragment carrying the full-length Tte polymerase gene was found, cloned, and sequenced. The primary structures of the Tte and Tth DNA polymerase genes were analyzed. The Tte-pol gene was recloned into an expression vector and recombinant protein was purified to homogeneity. The properties of Tte-pol in the polymerase chain reaction were investigated.     

Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element

Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.     

DNA sequence variation within the beta-glucuronidase gene complex among inbred strains of mice

Tightly linked to the gene that encodes murine beta-glucuronidase (GUS) are three GUS-specific regulatory elements. Together, these elements define the GUS gene complex. Specific alleles of each regulatory element are associated with a specific GUS structural allele. These associations define the three common forms (haplotypes) of the GUS gene complex, designated A, B, and H. As an initial step in defining the DNA determinants of each regulatory element and to develop DNA markers for the common haplotypes, we have identified several DNA variants by blot hybridization analysis of restricted genomic DNA using GUS-specific cDNA probes. Of 30 tested restriction endonucleases, 24 reveal DNA polymorphisms that distinguish B- and H-haplotype DNA from that of the A haplotype. Of these 24, 18 uncover a restriction fragment length polymorphism in which the polymorphic fragment of A-haplotype DNA is 200-300 bp larger than the corresponding fragment of B- or H-haplotype DNA. DNA sequence analysis of this polymorphic region reveals the presence of a short, interspersed repetitive element of the B2 family within A-haplotype DNA which is absent in DNAs of B- or H-haplotype mice. None of the DNA variations revealed by these analyses can be associated at this time with variation in the regulatory or structural properties of GUS among the common haplotypes. Nevertheless, they do provide useful haplotype-specific markers within the GUS gene complex which are of critical importance for DNA transfer experiments in transgenic mice and in cultured cells.     

Organization of chicken DNA sequences homologous to the transforming gene of avian myeloblastosis virus. II. Isolation and characterization of lambda proto-amv DNA recombinant clones from a library of leukemic chicken DNA.

Several lambda proto-amv recombinants isolated from a lambda Charon 4A library of leukemic chicken DNA were analyzed by using various restriction endonucleases and hybridization with specific probes representing different regions of the transforming gene of avian myeloblastosis virus. The position of 30 sites for 11 different restriction endonucleases was established in the proto-amv region of chicken DNA. Identical restriction endonuclease maps were obtained for the normal and leukemic DNAs in the proto-amv domain, which covers 8 to 9 kilobases of DNA. The cellular genetic elements homologous to the cellular sequence (amv) inserted into the avian myeloblastosis virus genome are contained within six major proto-amv segments which are interrupted by at least five large DNA regions lacking homology with amv.     

一种快速获得基因密码子偏爱性改造的方法-SOE-PCR

SOE-PCR采用具有互补末端的引物,使PCR产物形成重叠链,在不需要内切酶消化和连接酶处理的条件下实现DNA片段的拼接,能够高效、快速地实现基因的定点突变。应用SOE-PCR原理成功地实现了对几丁质酶基因chi58的5个位点的突变,构建了几丁质酶基因密码子偏爱性改造突变体-chi58A。实验证明,SOE-PCR具有简捷、快速、高效的优点。扩增过程中未引入其他突变,获得的chi58A突变体基因片段可以用于后续的分子克隆。     

Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.     

A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing

A set of plasmid vectors was developed which allows fast sequencing by the chemical degradation method. These high-copy-number vectors are derivatives of the plasmid pUC8 containing different multiple-purpose cloning sites flanked by unique recognition sequences for the restriction enzymes BstEII, Tth111I and Eco81I as sites for end-labelling DNA. Due to their partially asymmetric recognition sequences, each of these three restriction sites can be singly end-labelled by a filling-in reaction with selected nucleotides. This allows easy single end-labelling of any cloned DNA fragment for sequencing by the chemical degradation method without any isolation and purification step after the labelling reaction. In addition, the nucleotide sequence of the complementary strand from the same end can be determined by the dideoxy chain termination procedure using the universal M13 primers. In most of the new vectors, the reading frame of the lacZ' gene is retained, allowing identification of cloned fragments.     

Quikgene: a gene synthesis method integrated with ligation-free cloning

Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de novo synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics.     

几种新型植物基因表达载体的构建方法

利用基因工程技术手段研究基因功能过程中,构建基因表达载体处于转基因植物的主导地位,采用合适的构建方法会使实验效果事半功倍。植物基因表达载体的构建方法除了传统构建法、Gateway技术、三段T-DNA法、一步克隆法等,还有近年来出现的几种新型的载体构建方法:基于竞争性连接原理快速构建小片段基因表达载体;MicroRNA前体PCR置换法适用于构建小分子RNA表达载体;重组融合PCR法特别适用于插入片段中含有较多限制性酶切位点的载体构建;利用In-Fusion试剂盒可以将任何目的片段插入一个线性化载体的某个区域;构建多片段复杂载体可采用不依赖序列和连接的克隆方法(Sequence and ligation-independent cloning,SLIC)法;Gibson等温拼接法;Golden Gate拼接法。本文将在总结分析前人工作的基础上,结合自己工作的体会和经验分析这7种新方法的特点,期望通过这几种新的方法给植物基因工程表达载体的构建提供新的思路。     

Classification of rice germplasm. I. Analysis using ALP and PCR-based RFLP

The potential of using a PCR-based approach to detect DNA polymorphism for rice germplasm classification was compared with that of Southern-based RFLP analysis. Thirty-five Iranian rice varieties were studied along with 2 typical Indica and 3 typical Japonica varieties. Thirteen mapped RFLP markers were used as hybridization probes against Southern blots containing digests of one restriction endonuclease; 12 of the 13 probes detected polymorphism in the varieties. Fifteen sets of oligonucleotides derived from sequences near the ends of the same probes and of two other mapped probes were used as primers for PCR amplification of total genomic DNA of the varieties. Amplicon length polymorphisms (ALPs) were detected with 6 of the 15 sets of primers. To identify additional polymorphism, the PCR products were digested with nine different restriction endonucleases recognizing 4- or 5-bp DNA sequences and analyzed by gel electrophoresis in agarose and polyacrylamide. RFLPs were detected for 11 sets of primers, due to point mutations and to addition/deletion events that were too small to be detected as ALPs. Because PCR products are easily generated and may be analyzed in detail through the use of restriction endonucleases that cut rice DNA frequently, PCR-based RFLP analysis is a useful tool for the classification of rice germplasm.     

Generation of linear expression constructs by one-step PCR with vaccinia DNA topoisomerase I

Linear expression constructs can facilitate gene function studies. We describe a method to generate linear expression constructs for mammalian cells by one-step polymerase chain reaction (PCR) with vaccinia DNA topoisomerase I (TOPO). Cytomegalovirus (CMV) 5′ promoter, the gene of interest, and V5 bovine growth hormone (BGH) polyA 3′ terminator elements were PCR-amplified with target-specific primers containing vaccinia DNA TOPO-specific sequence and complementary sequence to each other. We amplified specific and complementary sequences. These three elements were directionally joined with vaccinia TOPO. The joined products were then directly transfected into Chinese hamster ovary cells. Compared with the transfection of supercoiled plasmids, comparable expression signals were obtained for green fluorescent protein, chloramphenicol acetyltransferase, and β-galactosidase proteins using Western blots. This is a quick and efficient method to generate linear expression construct. Unlike Invitrogen TOPO Tools, our method avoided the secondary round of PCR and more rapidly yielded correct joining products. This method can be easily used in the function test of uncharacterized open reading frames.     

Diversity of type II restriction endonucleases that require two DNA recognition sites

Orthodox Type IIP restriction endonucleases, which are commonly used in molecular biological work, recognize a single palindromic DNA recognition sequence and cleave within or near this sequence. Several new studies have reported on structural and biochemical peculiarities of restriction endonucleases that differ from the orthodox in that they require two copies of a particular DNA recognition sequence to cleave the DNA. These two sites requiring restriction endonucleases belong to different subtypes of Type II restriction endonucleases, namely Types IIE, IIF and IIS. We compare enzymes of these three types with regard to their DNA recognition and cleavage properties. The simultaneous recognition of two identical DNA sites by these restriction endonucleases ensures that single unmethylated recognition sites do not lead to chromosomal DNA cleavage, and might reflect evolutionary connections to other DNA processing proteins that specifically function with two sites.