Construction and validation of a Sinorhizobium meliloti whole genome DNA microarray: genome-wide profiling of osmoadaptive gene expression

Based on the complete Sinorhizobium meliloti genome sequence we established DNA microarrays as a comprehensive tool for systematic genome-wide gene expression analysis in S. meliloti 1021. For these PCR fragment-based microarrays, called Sm6kPCR, a collection of probes for the 6207 predicted protein-coding genes consisting of 6046 gene-specific PCR fragments and 161 70 mer oligonucleotides was arrayed in high density on glass slides. To obtain these PCR fragments primer pairs were designed to amplify internal gene-specific DNA fragments of 80-350 bp. Additionally, these primers were characterized by a 5' extension that allowed for reamplification using standard primers after the first amplification employing the specific primers. In order to ascertain the quality of the Sm6kPCR microarrays and to validate gene expression studies in S. meliloti parallel hybridizations based on RNA samples obtained from cells cultured under identical conditions were performed. In addition, gene expression in S. meliloti in response to an osmotic upshift imposed by the addition of 0.38 M NaCl was monitored. 137 genes were identified showing significant changes in gene expression resulting from the osmotic upshift. From these genes 52 were induced and 85 genes were repressed. Among the genes displaying different RNA levels some functional groups could be identified that are particularly remarkable. Repression was observed for 8 genes related to motility and chemotaxis, 7 genes encoding amino acid biosynthesis enzymes and 15 genes involved in iron uptake whereas 14 genes involved in transport of small molecules and 4 genes related to polysaccharide biosynthesis were induced.     

Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

Background

Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.

Results

We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.

Conclusion

A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.     

DNA analysis with multiplex microarray-enhanced PCR

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3′ end of a universal amplification primer. The complex primer pair is covalently tethered through its 5′ end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 10⁶-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 10³ genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.     

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.     

Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis

Background

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis.

Methods/Principal Findings

We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated.

Conclusions/Significance

Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.     

Comparative analysis of emm type pattern of Group A Streptococcus throat and skin isolates from India and their association with closely related SIC, a streptococcal virulence factor

Background

Recent studies showed that Helicobacter pylori existed in the New World prior to the arrival of Columbus. The purpose of the present study was to detect the presence of Helicobacter pylori in pre-Columbian mummies from Northern Mexico.

Methods

Six samples were studied (four samples of gastric remains, tongue-soft palate, and brain remained as negative controls) from two of the six naturally mummified corpses studied (adult male and infant male). Samples were taken from tissues suitable for DNA amplification by Polymerase chain reaction (PCR). DNA was extracted and H. pylori detection was carried out by PCR and hybridized with the pHp probe from 16S rRNA gene. The purified PCR products were cloned and sequenced in both directions. DNA sequences were analyzed with ALIGN and BLAST software. A second amplification was performed using ureB gene by real-time PCR.

Results

From four samples of gastric remnant, only two were H. pylori-positive for amplification of a 109 bp DNA fragment; the remaining two were negative, as were the tongue-soft palate and the brain biopsies as well. These PCR products were hybridized with a pHp probe. Nucleotide sequence analysis showed homology with H. pylori in 98 of 99% when compared with the gene bank nucleotide sequence. Only one sample of gastric remnant H. pylori-positive with 16S rRNA gene was also positive for ureB gene from H. pylori.

Conclusion

This data supported infection with H. pylori in Mexican pre-Columbian mummies dating from approximately 1,350 AC.     

LOMA: A fast method to generate efficient tagged-random primers despite amplification bias of random PCR on pathogens

Background  

Pathogen detection using DNA microarrays has the potential to become a fast and comprehensive diagnostics tool. However, since pathogen detection chips currently utilize random primers rather than specific primers for the RT-PCR step, bias inherent in random PCR amplification becomes a serious problem that causes large inaccuracies in hybridization signals.     

Highly parallel genome-wide expression analysis of single mammalian cells

Background

We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.

Methodology/Principal Findings

The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R²∼0.76–0.80 between individual cells and R²∼0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R²∼0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.

Conclusions/Significance

In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology.     

Genome-scale design of PCR primers and long oligomers for DNA microarrays

During the last years, the demand for custom-made cDNA chips/arrays as well as whole genome chips is increasing rapidly. The efficient selection of gene-specific primers/oligomers is of the utmost importance for the successful production of such chips. We developed GenomePRIDE, a highly flexible and scalable software for designing primers/oligomers for large-scale projects. The program is able to generate either long oligomers (40–70 bases), or PCR primers for the amplification of gene-specific DNA fragments of user-defined length. Additionally, primers can be designed in-frame in order to facilitate large-scale cloning into expression vectors. Furthermore, GenomePRIDE can be adapted to specific applications such as the generation of genomic amplicon arrays or the design of fragments specific for alternative splice isoforms. We tested the performance of GenomePRIDE on the entire genomes of Listeria monocytogenes (1584 gene-specific PCRs, 48 long oligomers) as well as of eukaryotes such as Schizosaccharomyces pombe (5006 gene-specific PCRs), and Drosophila melanogaster (21 306 gene-specific PCRs). With its computing speed of 1000 primer pairs per hour and a PCR amplification success of 99%, GenomePRIDE represents an extremely cost- and time-effective program.     

Including endophenotypes as covariates in variance component heritability and linkage analysis

Background

Simple and precise methods for sex determination in animals are a pre-requisite for a number of applications in animal production and forensics. However, some of the existing methods depend only on the detection of Y-chromosome specific sequences. Therefore, the abscence of a signal does not necessarily mean that the sample is of female origin, because experimental errors can also lead to negative results. Thus, the detection of Y- and X-chromosome specific sequences is advantageous.

Results

A novel method for sex identification in mammals (sheep, Ovis aries and European red deer, Cervus elaphus ) is described, using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. A partial sequence of the amelogenin gene of sheep and red deer was obtained, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. With a specific pair of primers a DNA fragment of different length between the male and female mammal was amplified.

Conclusion

PCR amplification using the amelogenin gene primers is useful in sex identification of samples from sheep and red deer and can be applied to DNA analysis of micro samples with small amounts of DNA such as hair roots as well as bones or embryo biopsies.     

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background

Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

Results

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

Conclusion

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.     

Template-blocking PCR: An advanced PCR technique for genome walking

This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3′ ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.     

Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wld s ) gene

Background

The strain of MeCP2^tm1.1Bird mice is a broadly used model for Rett syndrome. Because males carrying the invalidated MeCP2 locus are sterile, this strain has to be maintained in a heterozygous state. All animals therefore have to be genotyped at every generation to discriminate those carrying the invalidated allele (+/- females and y/- males) from those that do not. This is conveniently carried out by PCR on tail genomic DNA but because the primer pairs described initially for this purpose yield very similar size DNA bands on the WT and the KO alleles, this requires to carry out two independent PCR reactions on tail DNA preparations from all animals.

Results

After cloning and sequencing the PCR fragment amplified on the KO allele, we tested several sets of primers that were designed to yield PCR fragments of different sizes on the KO and WT alleles.

Conclusion

We have thus identified a set of three primers that allows for efficient genotyping of the animals by a single PCR reaction. Furthermore, using of this set of primers also resolves a recurrent problem related to the tendency of one of the initial primers to give rise to a non specific band because of its capacity to anneal at both ends of a repeated genomic element which we have identified as MurvyLTR.     

Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A,B, and D

Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.