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Matthew N Van Ert

Publications and source records attributed to Matthew N Van Ert.

10 recordsLinked to original sources
PubMed2006

Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain.

Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.

Anthrax↗
PubMed2005

Use of a real-time PCR TaqMan assay for rapid identification and differentiation of Burkholderia pseudomallei and Burkholderia mallei.

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.

Base Sequence↗
PubMed2005

Specific detection of bacillus anthracis using a TaqMan mismatch amplification mutation assay.

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation of Bacillus anthracis. The use of SNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene of B. anthracis. The assay permits specific, low-level detection (25 fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena of biodefense and microbial forensics.

Bacillus anthracis↗
PubMed2004

Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing.

Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B. anthracis, showing that it lies closer to a newly described "C" branch than to either of two previously described "A" or "B" branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.

Bacillus anthracis↗
PubMed2004

Cyclin mRNA stability does not vary during the cell cycle.

The cyclins are tightly regulated elements governing eukaryotic cell cycle progression by means of sequential activation-inactivation of cyclin-dependent kinases. In one manifestation of this regulation, the mRNA levels of several cyclin genes oscillate during the cycle in mammalian cells. Such cycle-dependent fluctuations in transcript levels could result from changes not only in rates of transcription, but also in mRNA stability. Here we used a new, minimally-disturbing method for producing multi-cycle synchronous growth of human MOLT-4 cells, in combination with quantitative real-time RT-PCR, to compare cell cycle-dependent transcript levels and half-lives of cyclin A2, B1, D3, E and PCNA mRNAs. While all mRNA levels except cyclin D3 varied in the cycle, there were no apparent variations in message half-lives. This differs from several previous reports of dramatic fluctuations in the stabilities of cyclin mRNAs, and infers that fluctuations in cyclin mRNA transcript levels during the MOLT-4 cell cycle are not due to variations in half-lives. The discrepancy in mRNA stability determinations could be due to differences in cell types or synchronization methods, but our findings may be representative of mRNA processing in the cycle of cells in unstressed steady-state growth.

Cell Cycle↗
PubMed2004

Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales.

Precise identification of Bacillus anthracis isolates has aided forensic and epidemiological analyses of natural anthrax cases, bioterrorism acts and industrial scale accidents by state-sponsored bioweapons programs. Because there is little molecular variation among B. anthracis isolates, identifying and using rare variation is crucial for precise strain identification. We think that mutation is the primary diversifying force in a clonal, recently emerged pathogen, such as B. anthracis, since mutation rate is correlated with diversity on a per locus basis. While single nucleotide polymorphisms (SNPs) are rare, their detection is facilitated by whole genome discovery approaches. As highly stable phylogenetic markers, SNPs are useful for identifying long branches or key phylogenetic positions. Selection of single, diagnostic "Canonical SNPs" (canSNPs) for these phylogenetic positions allows for efficient and defining assays. We have taken a nested hierarchal strategy for subtyping B. anthracis, which is consistent with traditional diagnostics and applicable to a wide range of pathogens. Progressive hierarchical resolving assays using nucleic acids (PHRANA) uses a progression of diagnostic genomic loci that are initially highly stable but with low resolution and, ultimately, very unstable but with high resolution. This approach mitigates the need for data weighting and provides both a deeply rooted phylogenetic hypothesis and high resolution discrimination among closely related isolates.

Animals↗
PubMed2004

Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis.

Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.

Alleles↗