A T-extended vector using a green fluorescent protein as an indicator

T-extended vector (T-vector) is a useful tool for cloning PCR products directly. We exploited a novel T-vector using a green fluorescent protein (GFP) as an indicator based on insertional inactivation. The brightest GFP mutant was used for easy detection even under daylight. The 100bp and 0.9kb of PCR products were cloned, and the transformant colonies with inserts were adjudged by the fluorescent green-white screening. The GFP system was more sensitive to insertional inactivation than the beta-galactosidase system at the conventional insertion sites.     

Identifying protein domains by global analysis of soluble fragment data

The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is challenging if there is little structural information concerning the protein target. One experimental procedure for mapping domains is to screen a library of random protein fragments for solubility, since truncation of a domain will typically expose hydrophobic groups, leading to poor fragment solubility. We have coupled fragment solubility screening with global data analysis to develop an effective method for identifying structural domains within a protein. A gene fragment library is generated using mechanical shearing, or by uracil doping of the gene and a uracil-specific enzymatic digest. A split green fluorescent protein (GFP) assay is used to screen the corresponding protein fragments for solubility when expressed in Escherichia coli. The soluble fragment data are then analyzed using two complementary approaches. Fragmentation “hotspots” indicate possible interdomain regions. Clustering algorithms are used to group related fragments, and concomitantly predict domain location. The effectiveness of this Domain Seeking procedure is demonstrated by application to the well-characterized human protein p85α.     

Use of tagged random hexamer amplification (TRHA) to clone and sequence minute quantities of DNA--application to a 180 kb plasmid isolated from Sphingomonas F199.

We have developed a novel method to clone and sequence minute quantities of DNA. The method was applied to sequence a 180 kb plasmid pNL1. The first step was the production of a size distributed population of DNA molecules that were derived from the 180 kb plasmid pNL1. The first step was accomplished by a random synthesis reaction using Klenow fragment and random hexamers tagged with a T7 primer at the primer 5'-end (T7-dN6, 5'-GTAATACGACTCACTATAGGGCNNNNNN-3'. In the second step, Klenow-synthesized molecules were amplified by PCR using T7 primer (5'-GTAATACGACTCACTATAGGGC-3'). With a hundred nanograms starting plasmid DNA from pNL1, we were able to generate Klenow-synthesized molecules with sizes ranging from 28 bp to >23 kb which were detectable on an agarose gel. The Klenow-synthesized molecules were then used as templates for standard PCR with T7 primer. PCR products of sizes ranging from 0.3 to 1.3 kb were obtained for cloning and sequencing. From the same Klenow-synthesized molecules, we were also able to generate PCR products with sizes up to 23 kb by long range PCR. A total 232.5 kb sequences were obtained from 593 plasmid clones and over twenty putative genes were identified. Sequences from these 593 clones were assembled into 62 contigs and 99 individual sequence fragments with a total unique sequence of 86.3 kb.     

Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs.

A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the BglII recognition sequence (AGATCT) in the GFPUV gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.     

Antibody binding loop insertions as diversity elements

In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.     

Cloning of anthozoan fluorescent protein genes

Many cnidarians display vivid fluorescence under proper lighting conditions. In general, these colors are due to the presence of fluorescent proteins similar to the green fluorescent protein (GFP) originally isolated from the hydrozoan medusa Aequorea victoria (Cnidaria: Hydrozoa). To optimize the search for new fluorescent proteins (FPs), a technique was developed that allows for the rapid cloning and screening of FP genes without the need for a prior knowledge of gene sequence. Using this method, four new FP genes were cloned, a green from Montastraea cavernosa (Anthozoa: Scleractinia: Faviidae), a cyan from Pocillopora damicornis (Anthozoa: Scleractinia: Pocilloporidae), a cyan from Discosoma striata (Anthozoa: Corallimorpharia), and a red from a second Discosoma species. Two additional green FPs were cloned, one from M. cavernosa and one from its congener Montastraea faveolata, from purified cDNA using PCR primers designed for the first M. cavernosa green FP. Each FP has recognizable amino acid sequence motifs that place them conclusively in the GFP protein family. Mutation of these products using a low-stringency PCR protocol followed by screening of large numbers of bacterial colonies allowed rapid creation of mutants with a variety of characteristics, including changes in color, maturation time, and brightness. An enhanced version of the new red FP, DspR1+, matures faster at 30 degrees C than the commercially available DsRed but matures slower than DsRed at 37 degrees C. One of the M. cavernosa green FPs, McaG2, is highly resistant to photobleaching and has a fluorescence quantum yield approximately twice that of EGFP-1.     

重要肠道病原菌多重PCR-基因芯片检测方法研究

目的:建立并初步评价一种针对重要肠道病原菌的多重PCR 基因芯片检测方法。方法：对筛选出的特异引物进行多重PCR优化,将引物分别按种属内混合和种属间混合的方案排查引物间的竞争性抑制现象,再将不同菌属的模板混合,用相对应的混合引物扩增,探寻高效特异的引物组合。分别掺入和不掺入荧光素,验证其对混合PCR反应的影响,并与芯片杂交,探寻多重PCR扩增效率对芯片杂交的影响。分析不同数量引物组合产生的杂交结果,筛选出无交叉反应的最优引物组合。结果:种属内引物混合均得到特异性扩增结果。种属间混合霍乱弧菌和空肠弯曲菌得到部分预期条带,随着混合引物数量的增加,交叉抑制现象也增多。杂交信号强度随多重PCR扩增效率的增加而增强。反应中掺入荧光素的扩增条带产量低于无荧光素的产物。可将35对混合引物拆成3个体系分别标记样品,以避免假阴性结果。结论:PCR反应中掺入荧光素降低扩增效率和杂交效率,但并不影响对杂交结果的判读和数据分析。基因芯片杂交信号强度取决于多重PCR的扩增效率。肠道病原菌多重PCR 基因芯片检测方法具有较高的特异性,混合PCR可以分别按照种属内和种属间的引物组合方案用于多病原的筛检。该基因芯片检测可以采用3个引物体系完成样品标记。     

Species identification of animal cells by nested PCR targeted to mitochondrial DNA

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.     

一种随机引物联合多特异性DNA片段检测结核分支杆菌的新方法

目的:针对目前结核性疾病实验室诊断的局限性,探索一种更为敏感和特异的结核分枝杆菌DNA检测新方法。方法:选取10株江苏地区流行的结核分支杆菌(MTB)菌株,选取临床其他常见菌株及分枝杆菌菌株作为对照组,分别提取DNA作为随机引物的模板。参考国内、外文献设计12条随机引物,并分别对MTB及对照菌株进行单个引物随机扩增,2%的琼脂糖凝胶电泳对扩增产物进行分离并切胶纯化,通过TA克隆将纯化片段连接到质粒pEASYTM-T5 Zero并进行测序,通过BLAST-nr比对验证是否为MTB DNA片段。按照所确定的MTB片段序列,在其内部设计、合成一对特异性引物。用此特异性引物扩增对应的随机引物扩增产物,获得MTB特异性条带图谱。并将该方法检测的敏感性和特异性与临床上常用的real-time PCR进行比较。结果:经BLAST-nr比对,随机引物IS986F,S535及IS986R扩增的条带与MTB DNA有高度同源性(均为99%)。随机引物IS986F、S535和IS986R分别联合其特异性引物可以检测稀释105倍、105倍和103倍的MTB DNA,其特异性分别为100%、90%和80%。常规real-time PCR可检测出稀释104倍的MTB DNA。结论:随机引物IS986F联合其特异性引物检测结核分枝杆菌的灵敏度和特异性优于S535、IS986R两组,特异性为100%,且灵敏度优于常规real-time PCR法。     

Computer analysis of band variability in DNA fingerprints

Through the example of the distribution of PCR products DNA matrices of mouse tail tissue, a method of comparative analysis of DNA fingerprints is described. The PCR products were obtained using a 20-mer random primer flanking the Atp1b2 locus on mouse chromosome 11. A software program was designed that permits the simplification of comparison of DNA fragments variability or polymorphism detected on electrophoregrams from different individuals.     

Hishot Display—A New Combinatorial Display for Obtaining Target-Recognizing Peptides

Display technologies are procedures used for isolating target-recognizing peptides without using immunized animals. In this study, we describe a new display method, named Hishot display, that uses Escherichia coli and an expression plasmid to isolate target-recognizing peptides. This display method is based on the formation, in bacteria, of complexes between a polyhistidine (His)-tagged peptide including random sequences and the peptide-encoding mRNA including an RNA aptamer against the His-tag. When this system was tested using a sequence encoding His-tagged green fluorescent protein that included an RNA aptamer against the His-tag, the collection of mRNA encoding the protein was dependent on the RNA aptamer. Using this display method and a synthetic library of surrogate single-chain variable fragments consisting of VpreB and Ig heavy-chain variable domains, it was possible to isolate clones that could specifically recognize a particular target (intelectin-1 or tumor necrosis factor-α). These clones were obtained as soluble proteins produced by E. coli, and the purified peptide clones recognizing intelectin-1 could be used as detectors for sandwich enzyme-linked immunosorbent assays. The Hishot display will be a useful method to add to the repertoire of display technologies.     

Microsatellite analysis using a two-step procedure for fluorescence labeling of PCR products.

A method for fluorescent labeling of PCR products has been developed. This method consists in a two-step procedure in which a first exponential classical PCR is followed by a "linear amplification". This second step relies on incorporation of fluorescent dNTP (dUTP or dCTP) in order to label the product on only one strand. The products can be applied without prior purification directly to a gel on a fluorescence-based automated DNA sequencer, for length and allele determination. The reliability of the results equals those of the classical 32P or fluorescent primer labeling methods, and the method is definitely less costly. Since the interpretation of the results is easier than with the method consisting in a fluorescent dNTP uptake in both strands in a single PCR, the present strategy should prove useful in mapping projects requiring analysis of a large number of microsatellites.     

Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR.

The PCR is used widely for the study of rRNA genes amplified from mixed microbial populations. These studies resemble quantitative applications of PCR in that the templates are mixtures of homologs and the relative abundance of amplicons is thought to provide some measure of the gene ratios in the starting mixture. Although such studies have established the presence of novel rRNA genes in many natural ecosystems, inferences about gene abundance have been limited by uncertainties about the relative efficiency of gene amplification in the PCR. To address this question, three rRNA gene standards were prepared by PCR, mixed in known proportions, and amplified a second time by using primer pairs in which one primer was labeled with a fluorescent nucleotide derivative. The PCR products were digested with restriction endonucleases, and the frequencies of genes in the products were determined by electrophoresis on an Applied Biosystems 373A automated DNA sequencer in Genescan mode. Mixtures of two templates amplified with the 519F-1406R primer pair yielded products in the predicted proportions. A second primer pair (27F-338R) resulted in strong bias towards 1:1 mixtures of genes in final products, regardless of the initial proportions of the templates. This bias was strongly dependent on the number of cycles of replication. The results fit a kinetic model in which the reannealing of genes progressively inhibits the formation of template-primer hybrids.     

An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation

A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.     

New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Sequence-tagged-site-facilitated PCR for barley genome mapping

Summary Speed, efficiency, and safety considerations have led many genome mapping projects to evaluate polymerase chain reaction (PCR) sequence amplification as an alternative to Southern blot analysis. However, the availability of informative primer sequences can be a limiting factor in PCR-based mapping. An alternative to random amplified polymorphism detection (RAPD) is the sequence-tagged-site (STS) approach. If informative primer sequences could be derived from known sequences, then current maps, which are based on both known function and anonymous clones, might be easily converted to maps utilizing PCR technology. In this paper, four pairs of primer sequences were obtained from published sequences, and four pairs were obtained by sequencing portions of DNA clones from genomic clones derived from a random genomic library used in the North American Barley Genome Mapping Project (NABGMP). These primers were used to screen for polymorphisms in the progeny of a winter x spring and a spring x spring barley cross. Two types of polymorphisms were distinguished using these primer sets: (1) insertion/deletion events that could be read directly from agarose gels, and (2) point mutation events. The latter were identified using polyacrylamide-gel electrophoresis of PCR products following digestion with restriction endonucleases (four-base cutters). To determine whether the PCR-based polymorphisms were allelic to polymorphisms identified by the clones from which the primer sequences derived, chromosomal assignments and (when possible) co-segregation analysis was performed.     

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

新型瞬时报告载体pRSET-EGFP的构建及表达

目的：利用分子生物学技术和方法将pRSET-B质粒改建为带有绿色荧光蛋白(GFP)突变体基因(GFP-S65T)的新型瞬时表达载体pRSET-EGFP,并在E．coli．BL21中得到GFP基因的高效表达。方法：PCR法从pEGFP质粒克隆GFP-S65T cDNA并在5’末端引入KpnⅠ的位点。将扩增出来的GFP-S65T基因和pRSEY-B质粒用HindⅢ和KpnⅠ双酶切后连接构成重组质粒。用化学法把重组质粒转化到E．coli．BL21中,培养发酵液。OD540=0．4时加入IPTG诱导GFP-S65T基因转录和表达,合成绿色荧光蛋白。还对诱导条件进行了优化,发酵液OD540=0．4加入IPTG可以得到最优表达。结果：通过Ni^2 柱亲和层析,纯化得到绿色荧光蛋白,SDS-PAGE电泳检测,分子量为27kDa,与文献报道值一致。这说明Ni^2 柱能够有效的纯化表达产物。结论：成功构建了新型瞬时表达载体pRSET-EGFP,并且在E．coli．BL21中得到高效表达。     

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.