Development and amplification of multiple co-dominant genetic markers from single spores of arbuscular mycorrhizal fungi by nested multiplex PCR

Multiple co-dominant genetic markers from single spores of the arbuscular mycorrhizal (AM) fungi Glomus mosseae, Glomus caledonium, and Glomus geosporum were amplified by nested multiplex PCR using a combination of primers for simultaneous amplification of five loci in one PCR. Subsequently, each marker was amplified separately in nested PCR using specific primers. Polymorphic loci within the three putative single copy genes GmFOX2, GmTOR2, and GmGIN1 were characterized by sequencing and single strand conformation polymorphisms (SSCP). Primers specific for the LSU rDNA D2 region were included in the multiplex PCR to ensure correct identification of the Glomus spp. spores. Single AM fungal spores were characterized as multilocus genotypes by combining alleles of each amplified locus. Only one copy of each putative single copy gene could be amplified from each spore, indicating that spores are homokaryotic. All isolates of G. mosseae had unique genotypes. The amplification of multiple co-dominant genetic markers from single spores by the nested multiplex PCR approach provides an important tool for future studies of AM fungi population genetics and evolution.     

A fast,highly sensitive double‐nested PCR‐based method to screen fish immunobiomes

Efficient methods for constructing 16S tag amplicon libraries for pyrosequencing are needed for the rapid and thorough screening of infectious bacterial diversity from host tissue samples. Here we have developed a double‐nested PCR methodology that generates 16S tag amplicon libraries from very small amounts of bacteria/host samples. This methodology was tested for 133 kidney samples from the lake whitefish Coregonus clupeaformis (Salmonidae) sampled in five different lake populations. The double‐nested PCR efficiency was compared with two other PCR strategies: single primer pair amplification and simple nested PCR. The double‐nested PCR was the only amplification strategy to provide highly specific amplification of bacterial DNA. The resulting 16S amplicon libraries were synthesized and pyrosequenced using 454 FLX technology to analyse the variation of pathogenic bacteria abundance. The proportion of the community sequenced was very high (Good’s coverage estimator; mean = 95.4%). Furthermore, there were no significant differences of sequence coverage among samples. Finally, the occurrence of chimeric amplicons was very low. Therefore, the double‐nested PCR approach provides a rapid, informative and cost‐effective method for screening fish immunobiomes and most likely applicable to other low‐density microbiomes as well.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.     

Improvements of PCR-based identification targeting the DNA topoisomerase II gene to determine major species of the opportunistic fungi Candida and Aspergillus fumigatus

For the simple and rapid detection/identification of major pathogenic fungal species such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and Aspergillus fumigatus, common primers for these species and specific primers for each species, designed on the basis on the genomic nucleotide sequences of the DNA topoisomerase II genes, were prepared and tested for their specificities in PCR amplifications. Twelve specific primers were pooled and designated PsVI. Genomic DNAs were amplified by the common primer pair, and followed by PCR amplification using PsVI. Using PsVI, six unique DNA fragments, all of which corresponded to a Candida or A. fumigatus species, were specifically and acceptably amplified from each template DNA even in the presence of other DNAs. Similarly, the results of identification of clinical samples based on the PCR amplification coincided with those of conventional identification techniques. The sensitivities of the direct PCR and the nested PCR using PsVI were found to be 1,000 and 50 yeast cells, respectively.     

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.     

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Survey of Mariner-Like Elements in the Housefly, Musca Domestica

The presence of mariner-like elements in four strains of the housefly, Musca domestica, was surveyed by PCR. Using the inverted terminal repeat (ITR) sequences of the Mos 1element as primers, DNAs were successfully amplified from all strains of the housefly. Southern blot analysis indicated that these amplified DNAs were repetitive sequences in the genome of M. domestica. Sequence analyses of cloned PCR products showed that they were 45% identical to the Mos 1element. These fragments appeared to be nonfunctional, because they contained no intact open reading frame (ORF) capable of encoding transposase. We conclude that these DNAs are degraded mariner-like elements (MLEs) in M. domestica. Because these endogenous MLEs in M. domesticado not encode any functional proteins, they probably would not affect the behavior of mariner-based vectors if such were introduced into this species as transformation vectors.     

A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores

 We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene. Accepted: 25 October 2000     

Could nested PCR be applicable for the study of microbial diversity?

PCR-based methods for rRNA gene analysis have been widely used to study diversity of microbiology. However, the analysis would be difficult when the DNA content in samples is too low to be amplified by conventional PCR. Nested PCR comes up with the advantage of higher sensitivity. It can detect target DNA at several-fold lower concentrations than conventional PCR. However, the amplification bias and factors that potentially affect measurement of sample diversity associated with nested PCR method has received little attention. Here, nested PCR was compared to reconditioning PCR which is based on conventional PCR and it would reduce the formation of heteroduplex. We investigated the use of both nested and reconditioning PCR methods to construct clone libraries of 16S rRNA genes from four swimming pool water samples. Abundances of OTUs (operational taxonomic units) were correlated between the libraries (r ² = 0.88, P < 0.0005), and some OTUs had equivalent abundances in the two libraries using the Chi-square test. Differences in taxonomic groups, as well as diversity and richness estimators, were compared by paired t-test and the Wilcoxon test, respectively. There were no significant differences between clone libraries using these two PCR methods. The results of ∫-Libshuff analysis suggested that nested PCR have no particular biases in revealing OTU diversity of a bacterial community. Thus, nested PCR produce congruent pictures with reconditioning PCR in the microbial community analysis.     

人源噬菌体抗体库的构建及抗VEGF抗体的初步筛选分析

应用噬菌体表面呈递技术构建人抗体组合文库 .筛选获得了结合血管内皮细胞生长因子( VEGF)的人噬菌体 Fab抗体 ,并对所获抗体的多样性进行了进一步分析 .从不同人群外周血淋巴细胞提取总 RNA,经反转录后采用家族特异性免疫球蛋白可变区基因引物与免疫球蛋白信肽序列引物 ,通过改变 PCR条件或半套式扩增分别获得全部亚型的轻、重链抗体 Fab段 ,并重组到噬粒载体 p Comb3H中 ,经电转化大肠杆菌 XL- 1 Blue,构建了 1 .5× 1 0 8完整组合抗体库 .利用 VEGF12 1对该库经过 4轮固相筛选后 ,获得 1 2个可与 VEGF特异结合的阳性克隆 .酶谱分析表明了所获抗体克隆的多样性 .为通过基因工程改造 ,进一步获得可用于临床的人源 VEGF抗体奠定了基础 .     

Cloning and expression of a haloacid dehalogenase fromPseudomonas sp. strain 19 S

A dehalogenase gene specifying the utilization of a variety of haloacids byPseudomonas sp. Strain 19S has been cloned and expressed inE. coli. Our cloning strategy employed specific amplification of a fragment homologous toPseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digestedPseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified inE. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.Abbreviations 2-CPA 2-chloropropionate - MCA monochloro acetate - IPTG isopropyl-1-thio--D-galactoside - NBT nitroblue tetrazolium salt - PCR polymerase chain reaction - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside - X-phosphate 5-bromo-4-chloro-3-indolyl phosphate     

Nested multiplex PCR——A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Reliable fusion PCR using Taq polymerases and pGEM-T easy vectors

We describe a reliable method for the production of fusion PCR products that can be used to transform the wild-type bacteria to replace target genes for mutagenesis studies. The relevant gene fragments and selective cassette are first amplified separately by PCR using primers that produce overlapping ends. As economic Taq DNA polymerase is disappointed in producing overlap ends due to adding an extra 3′-end ‘A’ base which potentially blocks the successful fusion of the amplified fragments, we use a new primer design strategy to overcome this disadvantage by introducing an additional ‘A’ base in the overlap primers. The amplified gene fragments were then separately cloned into a pGEM-T easy vector and re-amplified with the aid of a universal primer T7/SP6. This procedure enables performing nested PCR with the outmost primers in the fusion reaction to reliably splice the gene fragments into a single molecule with all sequences in the desired order.     

A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1

Background

Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution.

Methodology/Principal Findings

HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB) sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL) and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions.

Conclusions/Significance

Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).     

A PCR-based amplification method retaining the quantitative difference between two complex genomes

With the increasing emergence of genome-wide analysis technologies (including comparative genomic hybridization (CGH), expression profiling on microarrays, differential display (DD), subtractive hybridization, and representational difference analysis (RDA)), there is frequently a need to amplify entire genomes or cDNAs by PCR to obtain enough material for comparisons among target and control samples. A major problem with PCR is that amplification occurs in a nonlinear manner and reproducibility is influenced by stray impurities. As a result, when two complex DNA populations are amplified separately, the quantitative relationship between two genes after amplification is generally not the same as their relation before amplification. Here we describe balanced PCR, a procedure that faithfully retains the difference among corresponding amplified genes by using a simple principle. Two distinct genomic DNA samples are tagged with oligonucleotides containing both a common and a unique DNA sequence. The genomic DNA samples are pooled and amplified in a single PCR tube using the common DNA tag. By mixing the two genomes, PCR loses the ability to discriminate among the different alleles and the influence of impurities is eliminated. The PCR-amplified pooled samples can be separated using the DNA tag unique to each individual genomic DNA sample. The principle of this method has been validated with synthetic DNA, genomic DNA, and cDNA applied on microarrays. By removing the bias of PCR, this method allows a balanced amplification of allelic fragments from two complex DNAs even after three sequential rounds of PCR. This balanced PCR approach should allow genetic analysis in minute laser-microdissected tissues, paraffin-embedded archived material, or single cells.