Cloning and expression of the HIV protein in <Emphasis Type="Italic">Escherichia coli</Emphasis> cell-free system

Sixteen kinds of human immunodeficiency virus (HIV) target genes were cloned by polymerase chain reaction (PCR) amplification, and specific plasmids were constructed as the templates for the expression of these genes in the cell-free system. Similarly, the linear PCR templates of these genes for cell-free protein expression were also constructed by using two PCR amplification process. These different templates can be employed to biosynthesize HIV proteins in the cell-free system simultaneously and can be adapted for some high-throughput processes. HIV protease (P10) was performed as a target protein, and two different templates (plasmid and PCR product) were prepared and used for P10 expression in the Escherichia coli cell-free system. The target protein P10 was detected in sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels either by using a plasmid template or by a PCR template. These results are promising and helpful to develop a high throughput process for drug discovery.     

Purification and characterization of pea thioredoxin f expressed in Escherichia coli

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.     

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with<Emphasis Type="Italic">HMG I/Y</Emphasis> as an endogenous reference gene

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C _T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC _T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.     

A generally applicable assay for the quantification of inhibitory effects on PCR

PCR amplification of target genes from environmental DNA extracts can suffer from PCR inhibition, caused by co-extracted substances. No simple assay has been available to quantify this inhibition. Therefore, a generally applicable PCR inhibition-assay was developed, which allows determination of statistically significant inhibition of PCR. This information is important when documenting quality of DNA in environmental extracts used as template for PCR.     

A set of simple PCR markers converted from sequence specific RFLP markers on tomato chromosomes 9 to 12

A set of 24 simple PCR markers was generated for tomato chromosomes 9, 10, 11 and 12. Polymorphism was sought for between Lycopersicon esculentum and one of six other Lycopersicon species (L. parviflorum, L. cheesmanii, L. hirsutum, L. pennellii, L. peruvianum, and L. chilense). PCR primers, which were designed from mapped RFLP sequences, were used to amplify genomic DNA of the different species and the PCR amplification products were screened for polymorphism by testing restriction enzymes. With this approach, 24 (71%) of the 34 selected RFLPs were converted into simple PCR markers. By using a reference population, the map positions of these markers relative to the original RFLP markers were verified. These markers are locus specific and can be efficiently used for alignment of linkage maps, mapping target genes and marker assisted selection.     

Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli . We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.     

An improved PCR-based amplification of unknown homologous DNA sequences

The PCR primers used for cloning of evolutionary conserved genes or homologous DNA sequences are usually guessmer oligonucleotides. We introduce a simple way using Pfu polymerase to overcome possible PCR amplification failure because of 3'-end mismatches of guessed primers with the target DNA.     

A proportional analysis method using non-kinetic real-time PCR

Prompted by increasing interest in proportional analysis of genetic types, we developed a simple assay technique for determining the ratio of a specific target gene in the total genes that can be amplified with the same PCR primer. The key feature of this method is that the following two tasks are performed in a single-tube real-time PCR system: task 1, PCR amplification of the total genes including the target using a labeled PCR primer, with concurrent monitoring of the total copy number of the PCR product; task 2, detection of the signal of the target gene at each cycle of amplification, using a labeled nucleotide probe. In principle, the ratio of the target gene to the total genes is represented by the signal detected in 'task 2' at the cycle in which the PCR product reached a prescribed copy number (assessed by 'task 1').     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

Effect of DMSO on PCR of Porphyra yezoensis (Rhodophyta) gene

In order to improve the predictability ofresults of PCR with Porphyra yezoensisUeda genes, a study was made of possiblemodifications to the basic PCR protocol. DMSO used as an adjuvant considerablyincreased amplification efficiency andspecificity of PCR, the optimalconcentration being 5%. This protocolallowed for DNA templates with a high GCcontent to be amplified by PCR withoutproblem.     

Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing "universal" degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus.     

Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction

We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.     

Expression,Purification and Molecular Structure Modeling of Thioredoxin (Trx) and Thioredoxin Reductase (TrxR) from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.     

Cloning and expression of a novel lipase gene from Pseudomonas fluorescens B52

Here we describe an advanced polymerase chain reaction (PCR) technique, the compatible ends ligation inverse PCR (CELI-PCR) for chromosome walking. In CELI-PCR, several restriction enzymes, which produce compatible cohesive ends, were used to digest target DNA simultaneously or sequentially to produce DNA fragments of suitable size. DNA fragments were then easily circularized and PCR amplification could be carried out efficiently. The previous limitations of inverse PCR were overcome, such as unavailable restriction sites, poor template DNA circularization, and low amplification efficiency. Therefore, successive chromosome walking was performed successfully. Our work, isolating a 11,395-bp fragment from Gossypium hirsutum, was presented as an example to describe how CELI-PCR was carried out.     

Identification of uricase as a potential target of plant thioredoxin: Implication in the regulation of nodule development

During symbiotic nodule development in legume roots, early signaling events between host and rhizobia serve critical determinants for the proper onset of nodule morphogenesis, nitrogen fixation, and assimilation. Previously we isolated thioredoxin from soybean nodules as one of differentially expressed genes during nodulation and noted its positive role in nitrogen fixation. To identify the target proteins of thioredoxin in nodules, we used thioredoxin affinity chromatography followed by mass spectrometry. Nodulin-35, a subunit of uricase, was found to be a target of thioredoxin. Their interaction was confirmed by pull-down assay and by bimolecular fluorescent complementation. With an increased uricase activity observed also in the presence of thioredoxin, these results appear to implicate a novel role of thioredoxin in the regulation of enzyme activities involved in nodule development and nitrogen fixation.     

多重实时荧光PCR相对定量法快速诊断唐氏综合征

为了建立一种基于多重实时荧光相对定量PCR技术并应用之于唐氏综合征分子诊断, 选择21号染色体上唐氏综合征特异区域基因片段(DSCR3)为目的基因, 以12号染色体上的磷酸甘油醛脱氢酶基因(GAPDH)为参照基因, 设计合成两对引物以及分别以不同荧光标记的TaqMan探针, 在同一个反应管中进行扩增。以相对定量指标△CT值区分唐氏综合征患者与正常人。采用EB 病毒转化技术, 把唐氏综合征患者外周血B 淋巴细胞转化成永生淋巴母细胞系作为标准品。通过优化反应条件, 使得目的基因和参照基因的扩增效率基本一致, 接近100%, 模板浓度在3～300 ng/μL范围内, △CT值的变异系数小于15%, 浓度在30 ng/μL时, 变异系数最小(＜10%), 以该浓度的DNA作为模板进行批内和批间实验的△CT值重复性好, 变异系数分别为9.8%和13.3%。运用建立的方法检测20例唐氏综合征患者的血标本和30例正常人的血标本, 正常人△CT值范围是-1.90～-1.30, 患者的△CT值范围是-2.95～-2.15, 两组之间无交叉重叠, 有明显差异(P＜0.001)。唐氏综合征患者永生细胞系建系成功 ,染色体核型和DNA 分析表明建系前后遗传是稳定的。因此, 实时荧光定量PCR比较△CT值的相对定量法快速诊断唐氏综合征是可行的。     

高GC含量DNA模板的PCR扩增

目的：探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。     

A simple procedure for polymerase chain reaction of the PSBA gene in algae: application to the screening of mutations conferring atrazine resistance and discrimination of natural populations of Porphyra linearis

A simplified procedure is described for polymerase chain reaction (PCR) of a partial sequence (bp 601–893) of the plastid gene psbA in the rhodophyte Porphyra linearis and the diatoms Haslea ostreria and Skeletonema costatum. This procedure involves the use of all tissues of P. linearis and live cell suspensions of H. ostreria or S. costatum as DNA templates, without any further purification of DNA. As in the case of PCR with DNA extracts, a single major band of the expected size (292 bp) was obtained after PCR for the three species. Sequences of the amplified fragments were aligned, confirming that the amplified products were part of the psbA gene. The method was then used to screen mutations in partial psbA genes of 23 samples of P. linearis collected at four different stations along the mid-Atlantic coast of France. An alignment was obtained indicating the existence of mutations, though not in codons known for herbicide resistance. All mutations found were silent. However, genetic polymorphism discriminated between samples collected from two stations. The method employed allows rapid amplification of the herbicide target gene and simplifies the procedure for screening mutations or populations in algae. Its application to other genes and species is considered.     

Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.     

Target region amplification polymorphism: A novel marker technique for plant genotyping

The advent of large-scale DNA sequencing technology has generated a tremendous amount of sequence information for many important organisms. We have developed a rapid and efficient PCR-based technique, which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted candidate gene sequences. This target region amplification polymorphism (TRAP) technique uses 2 primers of 18 nucleotides to generate markers. One of the primers, the fixed primer, is designed from the targeted EST sequence in the database; the second primer, the arbitrary primer, is an arbitrary sequence with either an AT-or GC-rich core to anneal with an intron or exon, respectively. PCR amplification is run for the first 5 cycles with an annealing temperature of 35°C, followed by 35 cycles with an annealing temperature of 50°C. For different plant species, each PCR reaction can generate as many as 50 scorable fragments with sizes ranging from 50–900 bp when separated on a 6.5% polyacrylamide sequencing gel. The TRAP technique should be useful in genotyping germplasm collections and in tagging genes governing desirable agronomic traits of crop plants.