PyroMark® Instruments, Chemistry, and Software for Pyrosequencing® Analysis.

Since the early 2000s, Pyrosequencing(®) technology has been adapted for various instrument platforms to enable users to examine the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock, drug resistance development in pathogens, and polymorphisms in forensic samples of mitochondrial DNA.The instruments, software, and chemistry have been modified to facilitate different sample throughputs and sample amounts. Just recently, major changes have been implemented to enable increased read length and more precise Pyrosequencing results. These improvements were made possible through a number of changes to various system components. In addition, assay development has been streamlined through the availability of optimized PCR and Pyrosequencing reagents, automated assay design tools, and a number of predesigned Pyrosequencing assays.In future, instruments with smaller footprints and the ability to automate crucial steps of the Pyrosequencing protocol will be available and will provide even more convenient and standardized Pyrosequencing analysis with flexible throughput.

Pyrosequencing: powerful and quantitative sequencing technology.

Pyrosequencing is a sequencing-by-synthesis method for DNA analysis that has emerged as a platform not only for de novo sequencing applications, but also for quantitative analysis of genomic methylation, single-nucleotide polymorphisms, and allele quantification. In this unit, we describe a complete workflow from sample to result that is suitable for each of these applications. As cytosine conversion is a key element of successful methylation analysis using pyrosequencing, a support protocol for bisulfite treatment is also included.

A distinct DNA-methylation boundary in the 5'- upstream sequence of the FMR1 promoter binds nuclear proteins and is lost in fragile X syndrome.

We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.

Human CAR gene expression in nonpermissive hamster cells boosts entry of type 12 adenovirions and nuclear import of viral DNA.

Adenovirus type 12 (Ad12) propagation in hamster BHK21 cells is blocked prior to viral DNA replication. The amounts of Ad12 DNA in the nuclei or cytoplasm of hamster cells are about 2 orders of magnitude (2 h postinfection [p.i.]) and 4 to 5 orders of magnitude (48 h p.i.) lower than in permissive human cells. Cell line BHK21-hCAR is transgenic for and expresses the human coxsackie- and adenovirus receptor (hCAR) gene. Nuclear uptake of Ad12 DNA in BHK21-hCAR cells is markedly increased compared to that in naïve BHK21 cells. Ad12 elicits a cytopathic effect in BHK21-hCAR cells but not in BHK21 cells. Quantitative PCR or [(3)H]thymidine labeling followed by zone velocity sedimentation fails to detect Ad12 DNA replication in BHK21 or BHK21-hCAR cells. Newly assembled Ad12 virions cannot be detected. Thus, the block in Ad12 DNA replication in hamster cells is not released by enhanced nuclear import of Ad12 DNA.

Epigenetic status of an adenovirus type 12 transgenome upon long-term cultivation in hamster cells.

The epigenetic status of integrated adenovirus type 12 (Ad12) DNA in hamster cells cultivated for about 4 decades has been investigated. Cell line TR12, a fibroblastic revertant of the Ad12-transformed epitheloid hamster cell line T637 with 15 copies of integrated Ad12 DNA, carries one Ad12 DNA copy plus a 3.9-kbp fragment from a second copy. The cellular insertion site for the Ad12 integrate, identical in both cell lines, is a >5.2-kbp inverted DNA repeat. The Ad12 transgenome is packaged around nucleosomes. The cellular junction is more sensitive to micrococcal nuclease at Ad12-occupied sites than at unoccupied sites. Bisulfite sequencing reveals complete de novo methylation in most of the 1,634 CpGs of the integrated viral DNA, except for its termini. Isolated unmethylated CpGs extend over the entire Ad12 integrate. The fully methylated transgenome segments are characterized by promoter silencing and histone H3 and H4 hypoacetylation. Nevertheless, there is minimal transcriptional activity of the late viral genes controlled by the fully methylated major late promoter of Ad12 DNA.