Relative abundances of proteobacterial membrane-bound and periplasmic nitrate reductases in selected environments

Dissimilatory nitrate reduction is catalyzed by a membrane-bound and a periplasmic nitrate reductase. We set up a real-time PCR assay to quantify these two enzymes, using the narG and napA genes, encoding the catalytic subunits of the two types of nitrate reductases, as molecular markers. The narG and napA gene copy numbers in DNA extracted from 18 different environments showed high variations, with most numbers ranging from 2 x 10(2) to 6.8 x 10(4) copies per ng of DNA. This study provides evidence that, in soil samples, the number of proteobacteria carrying the napA gene is often as high as that of proteobacteria carrying the narG gene. The high correlation observed between narG and napA gene copy numbers in soils suggests that the ecological roles of the corresponding enzymes might be linked.     

Homolog-specific PCR primer design for profiling splice variants

To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org.     

Aerobic nitrate respiration in a nitrite-oxidising bioreactor

The ability of heterotrophic bacteria in a nitrite-oxidising bioreactor to respire with nitrate as an electron acceptor was examined. Approximately 70% of 1000 heterotrophic isolates were able to express a nitrate reductase. A detailed survey of 15 isolates showed that five expressed the azide-insensitive nitrate reductase encoded by the napA gene. A two-round PCR amplification of the napA gene using degenerate PCR primers and DNA sequence analysis of these products confirmed the presence of this gene in the positive isolates. Partial 16S rDNA products and napA products were amplified from the biomass in the bioreactor and denaturing gradient gel electrophoresis of these products identified 21 distinct ribotypes and 12 distinct napA sequences. The results show that the ability to respire with nitrate as an electron acceptor under aerobic conditions is widespread among the heterotrophic population of this bioreactor.     

Group‐specific primers for amplifying DNA sequences that identify Amphipoda,Cephalopoda, Echinodermata,Gastropoda, Isopoda,Ostracoda and Thoracica

We describe seven group‐specific primer pairs that amplify small sections of ribosomal RNA genes suitable for identification of animal groups of major importance as prey items in marine ecosystems. These primer sets allow the isolation of DNA from the target animal groups from mixed pools of DNA, where DNA‐based identification using universal primers is unlikely to succeed. The primers are designed for identifying prey in animal diets, but could be used in any situation where these animal groups are to be identified by their DNA.     

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

PCR primers that amplify fungal rRNA genes from environmental samples

Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.     

PCR detection of nearly any dengue virus strain using a highly sensitive primer 'cocktail'

PCR detection of viral pathogens is extremely useful, but suffers from the challenge of detecting the many variant strains of a given virus that arise over time. Here, we report the computational derivation and initial experimental testing of a combination of 10 PCR primers to be used in a single high-sensitivity mixed PCR reaction for the detection of dengue virus. Primer sequences were computed such that their probability of mispriming with human DNA is extremely low. A 'cocktail' of 10 primers was shown experimentally to be able to detect cDNA clones representing the four serotypes and dengue virus RNA spiked into total human whole blood RNA. Computationally, the primers are predicted to detect 95% of the 1688 dengue strains analyzed (with perfect primer match). Allowing up to one mismatch and one insertion per primer, the primer set detects 99% of strains. Primer sets from three previous studies have been compared with the present set of primers and their relative sensitivity for dengue virus is discussed. These results provide the formulation and demonstration of a mixed primer PCR reagent that may enable the detection of nearly any dengue strain irrespective of serotype, in a single PCR reaction, and illustrate an approach to the broad problem of detecting highly mutable RNA viruses.     

Primers to highly conserved elements optimized for qPCR‐based telomere length measurement in vertebrates

Telomere length dynamics are an established biomarker of health and ageing in animals. The study of telomeres in numerous species has been facilitated by methods to measure telomere length by real‐time quantitative PCR (qPCR). In this method, telomere length is determined by quantifying the amount of telomeric DNA repeats in a sample and normalizing this to the total amount of genomic DNA. This normalization requires the development of genomic reference primers suitable for qPCR, which remains challenging in nonmodel organism with genomes that have not been sequenced. Here we report reference primers that can be used in qPCR to measure telomere lengths in any vertebrate species. We designed primer pairs to amplify genetic elements that are highly conserved between evolutionarily distant taxa and tested them in species that span the vertebrate tree of life. We report five primer pairs that meet the specificity and reproducibility standards of qPCR. In addition, we demonstrate an approach to choose the best primers for a given species by testing the primers on multiple individuals within a species and then applying an established computational tool. These reference primers can facilitate qPCR‐based telomere length measurements in any vertebrate species of ecological or economic interest.     

EST derived PCR-based markers for functional gene homologues in cotton.

We investigated the utility of the Gossypium arboreum EST sequences in the GenBank database for developing PCR-based markers targeting known-function genes in cultivated tetraploid cottons, G. hirsutum and G. barbadense. Four hundred sixty-five randomly selected ESTs from this library were subjected to BLASTn search against all GenBank databases, of which putative function was assigned to 93 ESTs based on high nucleotide homology to previously studied genes. PCR primers were synthesized for 89 of the known-function ESTs. A total of 57 primer pairs amplified G. arboreum genomic DNA, but only 39 amplified in G. hirsutum and G. barbadense, suggesting that sequence divergence may be a factor causing non-amplification for some sites. DNA sequence analysis showed that most primer pairs were targeting the expected homologous loci. While the amplified products that were of larger size than the corresponding EST sequences contain introns, the primer pairs with a smaller amplicon than predicted from the flanking EST sequences did not amplify the expected orthologous gene sequences. Among the 39 primer pairs that amplified tetraploid cotton DNA, 3 detected amplicon size polymorphisms and 10 detected polymorphisms after digestion with one of six restriction enzymes. Ten of the polymorphic loci were subsequently mapped to an anchor RFLP map. Digestion of PCR-amplified sequences offers one means by which cotton genes can be mapped to their chromosomal locations more quickly and economically than by RFLP analysis.     

白菜的EST标记及其对油菜的通用性

根据白菜的表达序列标签,设计了28对引物。在对引物、dNTP、MgCl2的浓度及退火温度等参数进行测试后,建立了合适的PCR反应体系。在此反应体系下,以构建EST的白菜自交系A的DNA为模板,对设计的引物进行了筛选,发现有18对引物能对白菜DNA扩增出产物。用筛选出来的引物分别对17个白菜类品种进行PCR扩增,用琼脂糖凝胶电泳分析其产物的多态性,发现10对引物有多态性,这占了筛选引物的55.6%。为检测白菜EST标记的通用性,进一步利用设计的引物对不同油菜品种的DNA进行PCR扩增。在检测的28对引物中,共有24对引物能扩增出产物,占引物总数的85.7%,显示多态性的引物为18对,占引物总数的64.3%.。在对白菜DNA能扩增出产物的18对引物中,对油菜完全可用,且有13对引物产生多态性。而在那些对白菜未扩增出产物的10对引物中,也有6对能扩增出产物,其中5对显示多态性。文章研究结果证明,通过EST建立分子标记是可行的,而且这种标记对近缘物种是可通用的。     

Single- and double-SSR primer combined analyses in rice

Polymerase chain reaction (PCR) is the foundation of SSR molecular marker technology. We used sib rice varieties J518, XD1 and SD23 as experimental materials, selecting 30 pairs of SSR primers, including RM127, RM337 and RM5172, covering the rice genome, and performed single- and double-SSR primer combined analyses. We found that under the same PCR system and conditions, a single primer of the SSR primer pairs could amplify the same fragments as double primers do. The sequencing results demonstrated that some amplified fragments that we previously believed to come from double primers were actually produced by a single primer. The use of this kind of primer, such as the RM127 primer pair, for marker-assisted breeding will therefore be misleading. Additionally, using the same PCR system and conditions, some single primers that are part of SSR primer pairs can amplify many more specific fragments than double-SSR primers. For instance, in the case of the RM5172 primer pair, a single primer P1 amplified approximately three times the number of fragments as the double primer. This information can contribute to research on genetic diversity of species, understanding of genetic relationships and identification of germplasm resources. Accordingly, combined analyses of single- and double-primer amplification products not only can remove single-primer amplification fragments and false-positives from double-primer amplification products in order to improve test accuracy, but also can facilitate research on genetic diversity, exploration of phylogenetic relationships and identification of germplasm resources. We define this method as "single- and double-SSR primer combined analyses".     

PCR Primers That Amplify Fungal rRNA Genes from Environmental Samples

Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.     

Species and Strain Identification of the Predatory Mite Euseius Finlandicus by RAPD-PCR and ITS Sequences

The variation within and between Finnish Euseius finlandicus populations was investigated by RAPD-PCR and ITS sequence analyses. Resin DNA extraction was found to be a suitable method for samples of single mites used in PCR. The banding patterns from 24 RAPD primers and 10 primer pairs were very similar and reproducible in all specimens of the predatory mite studied. However, the E. finlandicus K-strain could be distinguished from organophosphate-resistant predatory mites (R-strain), since almost all of them produced a 1,400 bp RAPD-PCR product, which was missing or very rare in other strains studied. Another RAPD band of ca. 680 bp was in turn much more common in other mites of E. finlandicus than in the K-strain mites. Mite specific primers were designed and used to follow the survival of the R-strain released on apple trees. The 680 bp band obtained with specific primers was specific to the species E. finlandicus mites studied, including those that had been negative with RAPD primers. The 1,400 bp specific primers could be used as a marker for following the survival of R-strain mites on apple trees. At the species level it was possible to distinguish adults and eggs of E. finlandicus from Anthoseius rhenanus and Phytonemus pallidus by RAPD-PCR. In addition, a band at 480bp was found to correspond to DNA of the predatory mite Phytoseius macropilis, when both specific primer pairs were used together. It was not possible to amplify the ITS region of E. finlandicus rDNA using several primer pairs that work in other mites and aphids. However, a basidiomycete rDNA sequence was amplified with one of these ITS primer pairs in K-strain mites. Finally, it was found that fungal rDNA-specific primers amplified an ITS region of ca. 650 bp in several strains of E. finlandicus. Internal primers, designed to amplify the central part of the 650 bp product, successfully amplified this product from all the mites.     

Tetranucleotide,trinucleotide, and dinucleotide loci from the bobcat (Lynx rufus)

We describe primers and polymerase chain reaction (PCR) conditions to amplify four dinucleotide, one trinucleotide, and three tetranucleotide microsatellite DNA loci from the bobcat (Lynx rufus). The primers were tested on 22 individuals collected from a population located within southwestern Georgia (USA). The primer pairs developed in this study yielded an average of 7.4 alleles per locus (range four to 10), an average observed heterozygosity of 0.60 (range 0.40 to 0.76), and an average polymorphic information content of 0.70 (range 0.51 to 0.78).     

RAPD及SRAP两种分子标记技术对苦瓜杂交种纯度的鉴定分析

以F1代苦瓜杂交种如玉11号及其亲本为材料,利用RAPD及SRAP两种分子标记技术对这3种苦瓜基因组DNA进行比较分析,以获得该杂交种及其亲本（或母本）差异目的基因片段。经过多次对该3种苦瓜叶片DNA提取,PCR扩增及其PCR产物的琼脂糖凝胶电泳分析,在供试的46个RAPD引物及121对SRAP引物中,筛选出1个RAPD引物及1对SRAP引物能区分该苦瓜杂交种及其母本种子,通过进一步验证分析,证明该两种分子标记的特异引物可作为如玉11号苦瓜杂交种子的纯度鉴定之用。     

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.     

Assignment of 60 human ESTs in cattle

As part of the human genome study, large-scale cDNA sequencing has produced thousands of Expressed Sequence Tags (ESTs). Généthon has mapped in human 10,000 of these ESTs and has shown that the primers of about 1000 ESTs could amplify bovine DNA. In this work, we have analyzed 233 primer pairs provided by Genethon, to assign type I sequences to the bovine genome by using a hamster-bovine somatic cell hybrid panel. Among these 233 primer pairs, 109 gave a specific PCR product with bovine genomic DNA, but for 50% the size of the PCR product was the same in cattle and hamster, requiring SSCP analysis. Finally, 60 ESTs were assigned to the bovine genome, and among them 46 were found on the bovine chromosome expected from heterologous painting data between cattle and human. Received: 16 December 1999 / Accepted: 6 May 2000     

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.