We have developed an improved method for constructing a full-length cDNA library using small quantity of material by modifying the original oligo-capping method. In our devised method, total RNAs are used in sequential oligo-capping steps directly without preliminary mRNA purification. Using this method, we constructed full- length cDNA libraries from 100 microg of total RNA. These libraries contained 8x10(5) to 8x10(6) independent clones with average insert sizes of 2.0 kb. Moreover, the number of full-length cDNAs containing the translation initiation codon ATG in the constructed libraries was estimated to 60-70%. In addition, 54% of the known cDNAs had a longer 5' end than the corresponding genes in the public database. Our results show that the method can be effectively used to construct full-length enriched cDNA libraries, especially, if starting material is limited.
Base Sequence ; Cloning, Molecular/*methods ; *Gene Library ; Molecular Weight ; RNA/*chemistry/genetics/*isolation & purification

The authors reviewed the new technologies used for Panax genus research, including molecular identification technologies (especially for DNA barcoding), modern biotechnologies (e. g. the first generation and second generation sequencing technologies), and gene cloning and identification in this paper. These technologies have been successfully applied to species identification, transcriptome analysis, secondary metabolite biosynthetic pathway and the key enzyme function identification, indicating that the application of modern biotechnologies provide guarantee for the molecular identification of Panax genus. The application of modern biotechnologies also reveals the genetic information of transcriptome and functional genomics, and promotes the design of Panax plants genomic map. In summary, the application of the new technologies lay the foundation for clarifying the molecular mechanisms of ginsenoside biosynthesis and enforcing the in vitro synthesis of important natural products and new drugs in future.
Biotechnology ; methods ; Cloning, Molecular ; DNA Fingerprinting ; Ginsenosides ; biosynthesis ; Panax ; enzymology ; genetics ; metabolism ; Research Design

Gene cloning is one of the most important and widely used technologies in molecular biology research. Generally, DNA fragment is cut with restriction enzyme, and then the product is ligated to a linearized vector with complementary sticky end or blunt end by DNA-ligase. This traditional DNA cloning method requires compatible enzyme recognition sites existing in both PCR fragment and targeted vector. Several ligase-free methods have been established to avoid the using of restriction enzyme. However, those methods are time-consuming, labor-intensive and expensive. To overcome these shortcomings, we developed an Exonuclease III based DNA cloning method that takes only 30 minutes with high cloning efficiency and significant economic advantage. Therefore, this method is suitable for large-scale gene cloning.
Cloning, Molecular ; methods ; DNA ; chemistry ; DNA Restriction Enzymes ; Exodeoxyribonucleases ; chemistry ; Genetic Vectors ; Polymerase Chain Reaction

Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly y-linolenic acid. In this process, delta6-Fatty acid desaturase (D6D) plays a key role due to its enzymatic properties that catalyze the delta6 site dehydrogenation of precursor linoleic acid (18:2delta(9, 12) n-6) and a-linolenic acid (18:3delta(9, 12, 15) n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acids (HUFA) synthesis pathways. After we have isolated and cloned the gene coding delta6-fatty acid desaturase from Thamnidium elegans As3.2806 (GenBank accession number DQ099380), our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR I and Kpn I , respectively. Then we ligated the digested DNA with T4 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of delta6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4 kb from the EcoR I digested sample, in which a 1.3kb 5' upstream sequence (GenBank accession number DQ309425) of delta6-fatty acid desaturase gene containing several putative regulatory elements including TATA. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans detla6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3 kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans delta6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.
Base Sequence ; Cloning, Molecular ; DNA Primers ; Linoleoyl-CoA Desaturase ; genetics ; Molecular Sequence Data ; Mucorales ; enzymology ; genetics ; Polymerase Chain Reaction ; methods

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The results indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.
Base Sequence ; Cloning, Molecular ; DNA Primers ; DNA, Complementary ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; SARS Virus ; genetics

Based on the transcriptome database of Salvia miltiorrhiza, specific primers were designed to clone a full-length cDNA of ent-kaurene oxidase synthase (SmKOL) using the RACE strategy. ORF Finder was used to find the open reading frame of SmKOL cDNA, and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5.1. The transcription level of SmKOL from the hairy roots induced by elicitor methyl jasmonate (MeJA) was qualifiedby real-time quantitative PCR. The full length of SmKOL cDNA was of 1 884 bp nucleotides encoding 519 amino acids. The molecular weight of the SmKOL protein was about 58.88 kDa with isoelectric point (pI) of 7.62. Results of real-time quantitative PCR analyses indicated that the level of SmKOL mRNA expression in hairy roots was increased by elicitor oMeJA, and reached maximum in 36 h. The full-length cDNA of SmKOL was cloned from S. miltiorrhiza hairy root, which provides a target gene for further studies of its function, gibberellin biosynthesis and regulation of secondary metabolites.
Amino Acid Sequence ; Cloning, Molecular ; Computational Biology ; methods ; Cytochrome P-450 Enzyme System ; chemistry ; genetics ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protein Structure, Tertiary ; Salvia miltiorrhiza ; enzymology

Xenotransplantation of porcine organs has the potential to overcome the acute shortage of human tissues and organs for human transplantation. Swine represents an ideal source of such organs owing to their anatomical and physiological similarities to human besides their plentiful supply. However, this procedure is also associated with a number of safety issues related to zoonotic infections. Among such zoonotically important pathogens, porcine endogenous viruses (PERVs) represent the most concerned virus as they persist asymptomatically and show germline transmission in pigs. They belong to gamma retroviruses and are of three types viruses: A, B and C. In the present study, PCR based cloning was performed with chromosomal DNA extracted from miniature pigs to analyze the envelope gene of PERVs. Amplified PCR fragments of about 1.5 Kb, covering the partial env gene, were cloned into pCR2.1-TOPO vectors and sequenced. A total of 51 env clones were obtained from two miniature pigs, types M149 and T1111. Phylogenetic analysis of these genes revealed the presence of only PERV type A and B in the proportion of 45% and 55%, respectively. Among these, 9 clones had the correct open reading frame: eight were PERV type A and one PERV type B. Since both these PERV types are polytropic and have the capacity to infect human cells, our data raise a concern that proviral PERVs might have the potential to generate infectious viruses during or after xenotransplantation in humans.
Clone Cells ; Cloning, Molecular* ; Cloning, Organism ; DNA ; Genes, env ; Humans ; Methods ; Open Reading Frames ; Polymerase Chain Reaction ; Retroviridae ; Swine* ; Transplantation, Heterologous ; Zoonoses

Xenotransplantation of porcine organs has the potential to overcome the acute shortage of human tissues and organs for human transplantation. Swine represents an ideal source of such organs owing to their anatomical and physiological similarities to human besides their plentiful supply. However, this procedure is also associated with a number of safety issues related to zoonotic infections. Among such zoonotically important pathogens, porcine endogenous viruses (PERVs) represent the most concerned virus as they persist asymptomatically and show germline transmission in pigs. They belong to gamma retroviruses and are of three types viruses: A, B and C. In the present study, PCR based cloning was performed with chromosomal DNA extracted from miniature pigs to analyze the envelope gene of PERVs. Amplified PCR fragments of about 1.5 Kb, covering the partial env gene, were cloned into pCR2.1-TOPO vectors and sequenced. A total of 51 env clones were obtained from two miniature pigs, types M149 and T1111. Phylogenetic analysis of these genes revealed the presence of only PERV type A and B in the proportion of 45% and 55%, respectively. Among these, 9 clones had the correct open reading frame: eight were PERV type A and one PERV type B. Since both these PERV types are polytropic and have the capacity to infect human cells, our data raise a concern that proviral PERVs might have the potential to generate infectious viruses during or after xenotransplantation in humans.
Clone Cells ; Cloning, Molecular* ; Cloning, Organism ; DNA ; Genes, env ; Humans ; Methods ; Open Reading Frames ; Polymerase Chain Reaction ; Retroviridae ; Swine* ; Transplantation, Heterologous ; Zoonoses

After the cell enters into its programmed cell death, xylanases from grass plants gradually matured through its N-terminal and C-terminal sequence been cut by acid proteases several times. They could not be expressed by conventional protein expression system. Search the GenBank database, xynIII from a mutant of T. reesei QM9414(ATCC26921)was found. It is similar to grass plants' xylanase in their families and structures. It couldn't express in T. reesei QM9414, but its gene exist in genomic DNA as one copy. Through overlap-PCR method, 4 exons of xynIII were cloned, sequenced, spliced, and the whole cDNA of mature xynIII was acquired. The cDNA was inserted into pETBlue-2 vector and transformed into E. coli DE3 pLacI cell. Xyn III could be expressed in the transformed cell under the conditions of 37 degrees C, 1 mmol/L IPTG induced for 3h. Low temperature (15 degrees C), long time(64h) induction(0.2 mmol/L IPTG) could enhance xynIII activity.
Cloning, Molecular ; DNA, Complementary ; chemistry ; Endo-1,4-beta Xylanases ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Trichoderma ; genetics

Polyunsaturated fatty acids (PUFAs) including gamma-linolenic acid are valuable products because of their involvement in several aspects of human health care. GLA has been claimed to play a crucial role in development and prevention of some skin diseases, diabetes, reproductive disorder and others. At present, market demand for most gamma-linolenic acid is growing continually and current sources are inadequate for satisfying this demand due to the significant problems of low productivity, complex and expensive downstream process and unstable quality. Therefore, seeking for alternative sources are demanding. delta6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs, which catalyses the conversion of linoleic acid and alpha-linolenic acid to gamma-linolenic acid and stearidonic acid respectively. Unfortunately, the structure information on membrane desaturases is scarce because of the technical limitations in obtaining quantities of purified protein and the intrinsic difficulties in obtaining crystals from membrane proteins. With the isolation of the genes coding for delta6-fatty acid desaturase from various organisms, its characteristics will be elucidated gradually. Here we concisely reviewed the recent progress on studies of molecular biology including the cloning of delta6-fatty acid desaturase gene, structure and function, phylogeny and prospects of gene engineering application.
Cloning, Molecular ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Engineering ; methods ; Phylogeny ; gamma-Linolenic Acid ; biosynthesis