共查询到20条相似文献,搜索用时 15 毫秒
1.
N. BIERNE A. TANGUY M. FAURE B. FAURE E. DAVID I. BOUTET E. BOON N. QUERE S. PLOUVIEZ P. KEMPPAINEN D. JOLLIVET D. MORAGA P. BOUDRY P. DAVID 《Molecular ecology resources》2007,7(4):562-566
We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5′‐tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark–recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual‐based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high‐throughput method, providing the number of sequences exceeds the number of individuals initially amplified. 相似文献
2.
K. Vandepitte O. Honnay J. Mergeay P. Breyne I. Roldán‐Ruiz T. De Meyer 《Molecular ecology resources》2013,13(2):269-275
Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost‐effective approaches to uncover genome‐wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD‐PE (Restriction site Associated DNA Paired‐End sequencing) approach. RAD tags were generated from the PstI‐digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired‐end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N50 = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD‐PE as an inexpensive genome‐wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers. 相似文献
3.
Mei-Qin Liu Xin Shen Wei-Lun Yin Cun-Fu Lu 《植物学报(英文版)》2007,49(3):382-385
T-A cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3' thymldylate residue at each of Its 3' termini to clone polymerase chain reaction (PCR)-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5' end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5' terminus respectively. The data shows that when the 5' end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5' cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3'dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products. 相似文献
4.
A 1330-bp DNA sequence with two XcmI cassettes was inserted into pUC18 to construct an efficient XcmI T-vector parent plasmid, pYEMF. The large size of the inserted DNA fragment improved T-vector cleavage efficiency, and guaranteed
good separation of the molecular components after restriction digestion. The pYEMF-T-vector generated from parent plasmid
pYEMF permits blue/white colony screening; cloning efficiency analysis showed that most white colonies (>75%) were putative
transformants which carried the cloning product. The sequence analysis and design approach presented here will facilitate
applications in the fields of molecular biology and genetic engineering. 相似文献
5.
Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability
to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower
temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed
π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway.
Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816 bp fragment of TIM gene which covers the 756 bp open reading frame was achieved by a combination
of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers
and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM
was 97% identical to TIM from Pseudomonas
fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The
cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction
and screening method. Homology model of the TIM protein in this study was generated based on Escherichia
coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further
investigation into areas that showed deviations from the known mesophilic TIM structures. 相似文献
6.
PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories. 相似文献
7.
Wei Wang Xiaofeng Ji Cui Yuan Fangqun Dai Jiancheng Zhu Mi Sun 《Indian journal of microbiology》2013,53(4):477-481
Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater. 相似文献
8.
Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using
another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling
for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%)
and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes. 相似文献
9.
Ingerid J. Hagen Anna M. Billing Bernt Rønning Sindre A. Pedersen Henrik Pärn Jon Slate Henrik Jensen 《Molecular ecology resources》2013,13(3):429-439
With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non‐model species. Here, we describe a successful approach to a genome‐wide medium density Single Nucleotide Polymorphism (SNP) panel in a non‐model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP‐chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP‐chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP‐chip to demonstrate the ability of such genome‐wide marker data to detect population sub‐division, and compared these results to similar analyses using microsatellites. The SNP‐chip will be used to map Quantitative Trait Loci (QTL) for fitness‐related phenotypic traits in natural populations. 相似文献
10.
W. Yang L. Han M. Mandlaa H. Chen M. Jiang Z. Zhang H. Xu 《Letters in applied microbiology》2013,57(1):54-62
Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l?1 h?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m3 fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.
Significance and Impact of the Study
KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening. 相似文献11.
Recovery of DNA barcode sequences is often challenging from the archived specimens. However, short fragments of DNA may be recovered, which would significantly improve many unresolved taxonomic conflicts. Here, we designed a mini‐barcode for catfishes comprising several species and many cryptic taxa. We analysed a data set of 3048 publicly available COI barcode sequences representing 547 worldwide catfish species and performed 152 628 interspecies comparisons. A significantly more positively correlated interspecies distance was detected with transversion (0.78, P < 0.001) than with transition (0.70, P < 0.001). This suggested that transversions were better diagnostics for species identification. In the aligned data set, two transversion‐rich fragments (53 bp and 119 bp) were identified. Transition/transversion bias value was 1.04 in 53‐bp fragment, 1.23 in 119‐bp fragment and 1.50 in full‐length barcode. The interspecies distance with full‐length barcode was 0.212 ± 0.037, while that with 53‐bp and 119‐bp fragments was 0.325 ± 0.039 and 0.218 ± 0.045, respectively. Survey of 53‐bp fragment showed a possibility of only 1144 barcodes, while that of 119‐bp fragment showed >4 million barcodes. Thus, the 119‐bp fragment is a viable mini‐barcode for catfishes comprising >3000 extant species. Experiment with 82 archived catfishes showed successful recovery of this mini‐barcode using the designed primer. The mini‐barcode sequences showed species‐specific similarity in the range of 98‐100% with the global database. Therefore, survey of a transversion‐rich fragment within the full‐length barcode would be an ideal approach of mini‐barcode design for biodiversity assessment. 相似文献
12.
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. 相似文献
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15.
Restriction fragment length analysis of PCR amplified 16S rDNA with AluI revealed the presence of a 265 bp fragment in all species of Bacillus with the exception of B. cereus and B. thuringiensis, which contains two restriction sites within this fragment which results in three smaller fragments totalling to 265 bp.
Some distant species of Bacillus with no evidence of this fragment could be delineated into other genera based on phenotypic and genotypic parameters. BLAST
search for homologous sequences of individual species revealed that it is a highly conserved region. Multiple alignment of
the fragment suggests that a region between 160 and 265 bp of the 265 bp fragment was a hypervariable region and were highly
species-specific. A set of primers was designed for amplification of this hypervariable region. Partial sequencing of the
hypervariable region within the 265 bp fragment seems an index for identification of Bacillus species. 相似文献
16.
Chalcone synthase is the key enzyme in biosynthesis of flavonoids, which play roles in pigmentation of flowers and protection
against ultraviolet and pathogens. Inverse polymerase chain reaction (IPCR) is a method for the rapid in vitro amplification
of DNA sequences that flank a region of known sequence. In this study, IPCR united with nested PCR was successfully applied
in cloning full-length sequences of three Phalaenopsis chalcone synthase genes (phchs3, phchs4, and phchs5, respectively). Firstly, routine PCR with homologous primers were performed, and gene fragments of phchs3 (1 kb), phchs4 (1.2 kb), and phchs5 (800 bp) were obtained and then sequenced. Then, inverse PCR were carried out for cloning full-length sequence of each gene.
Because products were not unique in single round inverse PCR, nested PCR were performed, and the specificity was much enhanced.
At last, full-length sequences of 2,499 bp for phchs3, 2,502 bp for phchs4, and 1,855 bp for phchs5 were obtained. This study proved that IPCR could be more efficient if being united with nested PCR. 相似文献
17.
18.
Sampali Banerjee Jitendra Kumar Anjali Apte-Deshpande Sriram Padmanabhan 《Microbial cell factories》2010,9(1):30
Background
The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc. 相似文献19.
Chen SL Li J Deng SP Tian YS Wang QY Zhuang ZM Sha ZX Xu JY 《Marine biotechnology (New York, N.Y.)》2007,9(2):273-280
The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish
sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of
28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer
combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and
males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464,
CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels,
cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain
reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers
was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females
produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also
investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about
350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular
sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth
tongue sole. 相似文献
20.
J. M. Pujolar M. W. Jacobsen T. D. Als J. Frydenberg K. Munch B. Jónsson J. B. Jian L. Cheng G. E. Maes L. Bernatchez M. M. Hansen 《Molecular ecology》2014,23(10):2514-2528
Next‐generation sequencing and the collection of genome‐wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD‐sequenced European eel individuals (glass eels) from eight locations between 34 and 64oN, we examined the patterns of genome‐wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using FST‐based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (FST = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single‐generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand–receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation. 相似文献