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  • PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. 7891182

    Neuronal plasticity associated with both short- and long-term administration of psychomotor stimulants involves alterations in specific patterns of gene expression. In order to screen for brain region specific mRNAs which are transcriptionally regulated by acute cocaine and amphetamine, PCR differential display was employed. This approach identified a previously uncharacterized mRNA whose relative levels in the striatum are induced four- to fivefold by acute psychomotor stimulant administration. Isolation and characterization of corresponding cDNA clones resulted in complete nucleotide sequence analysis, including prediction of the encoded protein product. Alternate polyA site utilization in the predicted 3' noncoding region results in the appearance of an RNA doublet, approximately 700 and 900 bases in length, following Northern analysis. A presumed alternate splicing event further generates diversity within the transcripts, and results in the presence or absence of an in-frame 39 base insert within the putative protein coding region. As a result, the predicted translation products are either 129 or 116 amino acids in length. A common hydrophobic leader sequence at the amino terminus is present within each predicted polypeptide, suggesting that the protein product is targeted for entry into the secretory pathway. Basal expression of the RNA doublet is limited to neuroendocrine tissues, further implying that the protein product plays a functional role in both neuronal and endocrine tissues.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Direct chromatin PCR (DC-PCR): hypotonic conditions allow differentiation of chromatin states during thermal cycling. 22984542

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents 16632318

    In a real-time PCR assay of Legionella pneumophila (targeting the L. pneumophila-specific mip gene and using SYBR Green dye for DNA detection in conjunction with the iCycler system) we detected as few as 1.3 copies of a mip gene in a 50-ml reaction from serially diluted L. pneumophila genomic DNA. However, cycle threshold (CT) were yielded and DNA product detected in our no-template negative controls and the phenomenon persisted when two separate batches of PCR reagents and water from two different biochemical companies were tested. Since L. pneumophila can be widespread in municipal water supplies, the commercial reagents, especially the reagent water (80% of the reaction volume), could be the source of contamination. To test this hypothesis, we treated Millipore Milli-Q water by filtering through a 0.2 mm-pore-size polycarbonate filter to remove bacteria prior to autoclaving. Real-time PCR using this water had no contamination. Our finding is indirect evidence that commercially available purified water can harbor low level contamination by L. pneumophila DNA that has escaped purification processes. This presents a challenge when developing a sensitive DNA-based bacterial detection method if the target organism or its DNA is a common contaminant of necessary reagents.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Closed-tube PCR methods for locus-specific DNA methylation analysis. 21913071

    Closed-tube PCR methods (sometimes referred to as in-tube PCR methods) for locus-specific DNA -methylation analysis are methodologies in which the amplification and analysis of bisulphite-modified DNA take place in one tube without the need to remove the PCR products for further analysis. Closed-tube methodologies lend themselves to high-throughput applications and molecular diagnostics but are also applicable as a research tool. We review three closed-tube methodologies, methylation-sensitive high-resolution melting (MS-HRM), MethyLight, and sensitive melting after real-time analysis - methylation-specific PCR (SMART-MSP). Closed-tube detection can be performed by simultaneously amplifying both methylated and unmethylated templates and subsequent melting curve analysis (MS-HRM). Alternatively, methylation-specific primers are used in real-time quantitative PCR and monitored either by a fluorescent hydrolysis probe (MethyLight) or using a double-stranded DNA binding fluorescent dye with a subsequent quality control step by melting curve analysis (SMART-MSP).
    Document Type:
    Reference
    Product Catalog Number:
    S7821
    Product Catalog Name:
    CpGenome Universal Methylated DNA