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Am. J. Bot. 97, 1574–8. Clonal structure of wild populations and origins of horticultural stocks of Illicium parviflorum (Illiciaceae). 2010

Newell, D.L. and Morris, A.B.

Notes: The authors investigated genetic diversity in a Florida population of Illicium parviflorum, an endangered evergreen shrub, by amplifying intersimple sequence repeats (ISSRs). Amplifications were performed using GoTaq® Hot Start Polymerase. (4161)

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Appl. Environ. Microbiol. 76, 3863–8. Comparison of normalization methods for construction or large multiplex amplicon pools for next-generation sequencing 2010

Harris, J.K., Sahl, J.W., Castoe, T.A., Wagner, B.D., Pollack, D.D. and Spear, J.R.

Notes: GoTaq® Master Mix was used for PCR of bacterial ribosomal RNA genes prior to pyrosequencing. (4529)

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Genetics 184, 119–28. Detection, validation, and downstream analysis of allelic variation in gene expression. 2010

Ciobanu, D.C., Lu, L., Mozhui, K., Wang, X., Jagalur, M., Morris, J.A., Taylor, W.L., Dietz, K., Simon, P. and Williams, R.W.

Notes: Sequence variation within a gene, such as single nucleotide polymorphisms (SNPs), can lead to differences in expressions levels of corresponding mRNAs; genes that are self-regulated by this mechanism (cis modulation) are difficult to identify accurately with existing techniques. The authors used bioinformatic and molecular approaches to estimate error rates when identifying cis-modulated transcripts and developed a simple method to detect these transcripts in C57BL/6J F1 hybrid mice. This method, which they named allelic specific expression, is RT-PCR-based and uses PCR primers that flank an informative SNP to quantify the differential expression levels of transcripts. GoTaq® Flexi DNA Polymerase was used in the PCR. (4051)

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Am. J. Pathol. 177, 2347–56. Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma. 2010

de Maat, M.F., Narita, N., Benard, A., Yoshimura, T., Kuo, C., Tollenaar, R.A., de Miranda, N.F., Turner, R.R., van de Velde, C.J., Morreau, H. and Hoon, D.S.

Notes: The authors examined the methylation status of methylated-in-tumor (MINT) loci and the degree of microsatellite instability (MSI) in colorectal cancer to determine if methylation of MINT loci during the progression of adenoma to cancer was linked to MSI. They used on-slide sodium bisulfite modification and methylation-specific PCR to examine the methylation index in paraffin-embedded tissue blocks containing normal, adenoma and cancer tissues. MSI status was determined using the MSI Analysis System. Patients with instability at more than 4 markers were classified as MSI-high, and patients with instability at more than 1 marker were considered microsatellite-stable. (4106)

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J. Biomol. Scr. 15, 418–26. Epitope mapping of antibodies using a cell array-based polypeptide library. 2010

Maier, R.H., Maier, C.J., Rid, R., Hintner, H., Bauer, J.W. and Onder, K.

Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

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Appl. Environ. Microbiol. 76, 3590–9. Growth of bacteria on 3-nitropropionic acid as a sole source of carbon, nitrogen, and energy. 2010

Nishino, S.F., Shin, K.A., Payne, R.B. and Spain, J.C.

Notes: The authors identified a bacterial strain that can use the toxin 3-nitropropionic acid (3NPA) as its sole carbon and nitrogen sources and cloned the genes that encode the enzymes responsible for the initial steps in the 3NPA degradation pathway. The Pseudomonas library used to clone these genes was created using genomic DNA isolated using the Wizard® SV Genomic DNA Purification System. Closely related genes were amplified from other bacterial species for phylogenetic analysis. PCRs were performed using the GoTaq® Hot Start Polymerase. (4164)

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Clin. Can. Res. 16, 1391–401. High-resolution array comparative genomic hybridization in sporadic and celiac disease-related small bowel adenocarcinomas. 2010

Diosdado, B., Buffart, T.E., Watkins, R., Carvalho, B., Ylstra, B., Tijssen, M., Bolijn, A.S., Lewis, F., Maude, K., Verbeke, C., Nagtegaal, I.D., Grabsch, H., Mulder, C.J., Quirke, P., Howdle, P. and Meijer, G.A.

Notes: To better examine the molecular mechanisms of small bowel adenocarcinomas, DNA was extracted from paraffin-embedded tissue and tested for microsatellite instability (MSI) using the MSI Analysis System, Version 1.2. The PCR products were run on the Applied Biosystems 3130 Genetic Analyzer and analyzed using GeneScan® software. If two or more of the BAT-25, BAT-26, NR-21, NR-24 or MONO-27 monomorphic markers had altered lengths, the tumors were designated as MSI unstable. (4112)

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Appl. Environ. Microbiol. 76, 829–42. Lysogeny and sporulation in Bacillus isolates from the Gulf of Mexico. 2010

Mobberley, J., Authement, R.N., Segall, A.M., Edwards, R.A., Slepecky, R.A. and Paul, J.H.

Notes: Certain bacteriophages can promote host cell sporulation under unfavorable conditions to increase survival of the host and prophage. These types of phages, known as spore-converting phages, have been found in terrestrial Bacillus species. In this article the authors examined the effect of prophages on sporulation of 11 Bacillus isolates from the Gulf of Mexico. Potential prophages in the Bacillus isolates were detected by PCR using unique PCR primer sets for each prophage genome and GoTaq® Green Master Mix. One of these isolates, B14905, was examined in more detail; the genome of this isolate was isolated using the Wizard® Genomic DNA Purification Kit, then sequenced. (4091)

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Vet. Pathol. 47, 163–6. Peliosis hepatis in cats is not associated with Bartonella henselae infections. 2010

Buchmann, A.U., Kempf, V.A., Kershaw, O. and Gruber, A.D.

Notes: The vasculoproliferative disorder peliosis hepatis has been linked to Bartonella henselae infection in humans and dogs. The authors sought to determine if this is true in cats, the natural reservoir for B. henselae, using immunohistochemistry (IHC) and PCR. Tissue sections from 26 cats with peliosis hepatis were formalin-fixed and paraffin-embedded and subjected to IHC using a B. henselae-specific antibody. To detect B. henselae DNA, PCR was performed using GoTaq® Flexi DNA Polymerase, genus-specific primers for the pap31 gene of Bartonella, 1mM MgCl2 and total DNA isolated from 8µm paraffin-embedded tissue sections. The presence of Bartonella DNA was confirmed by PCR using species-specific primers that target the B. henselae heat-shock protein (htrA). These studies found no link between B. henselae infection and peliosis heptis in cats. (4094)

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J. Exp. Bot. 61, 191–202. Physiological and molecular changes in Oryza meridionalis Ng., a heat-tolerant species of wild rice. 2010

Scafaro, A.P., Haynes, P.A. and Atwell, B.J.

Notes: The authors compared seedling growth rates, photosynthesis rates and expression levels of heat-responsive genes in the heat-resistant wild rice Oryza meridionalis and the domesticated rice O. sativa when grown at optimal and elevated temperatures. Proteins that were up- or downregulated in response to heat were identified by two-dimensional gel electrophoresis coupled with nano liquid chromatography on line with tandem mass spectrometry (nanoLC-MS/MS). Trypsin was used to cleave proteins prior to nanoLC-MS/MS. Semi-quantitative RT-PCR was performed using the GoTaq® Green Master Mix to determine if the heat-responsive proteins were transcriptionally regulated. (4092)

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J. Bacteriol. 192, 4720–31. Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function. 2010

Yun, C.S., Suzuki, C., Naito, K., Takeda, T., Takahashi, Y., Sai, F., Terabayashi, T., Miyakoshi, M., Shintani, M., Nishida, H., Yamane, H. and Nojiri, H.

Notes: The authors investigated the expression of genes encoding histone-like (H-NS) proteins from the self-transmissible pCAR1 plasmid and Pseudomonas putida KT2440 genome, as well as the interaction of H-NS family members in vitro. Gene expression was quantified using quantitative RT-PCR and RNA templates that were treated with RQ1 RNase-Free DNase to degrade contaminating DNA. Interactions between Pmr and other H-NS proteins were monitored using pull-down assays. His-tagged Pmr was expressed in E. coli, purified and used as bait for FLAG-tagged H-NS family proteins. Protein purification of His-tagged proteins was performed using the MagneHis™ Protein Purification System. (4119)

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RNA 16, 239–50. Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression. 2010

Walters, R.W., Bradrick, S.S. and Gromeier, M.

Notes: The authors investigated the mechanism of microRNA (miRNA)-mediated regulation of both endogenous mRNAs and artificial reporter constructs. To determine whether an m7G cap and poly(A) tail are required for repression, the authors created plasmids containing eight synthetic, tandem miR-30 recognition sequences in the 3´ untranslated region (UTR) of a Renilla luciferase gene under the control of a 5´UTR that confers either cap-dependent or cap-independent translation. They used these plasmids as a template for in vitro transcription, then transfected in vitro transcripts with and without an m7G cap and poly(A) tail into 293T cells, along with miR-30 miRNA (and miR-21 as a negative control). Similar experiments were performed by transfecting 293T cell with the Renilla luciferase constructs and miR-30 and miR-21 expression vectors. Finally, the authors exchanged the artificial 3´ UTR of the Renilla luciferase construct with the 3´ UTR of BACH1, a transcription factor that is regulated by miR-155, and cotransfected 293T cells with the BACH1 3´ UTR construct and an miR-115 expression vector to examine regulation via an endogenous 3´ UTR. The Promega Primer Extension System was used to compare miR-21, miR-30 and miR-155 RNA levels in transfected and untransfected 293T cells to determine endogenous levels of these miRNAs. Renilla luciferase activity was determined using the Renilla Luciferase Assay System. The authors used Northern blot analysis and quantitative RT-PCR to determine Renilla luciferase RNA levels (and GAPDH levels for normalization purposes). The Plexor® One-Step qRT-PCR System was used for qRT-PCR. (4048)

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Appl. Environ. Microbiol. 76, 2783–90. Revelation by single-nucleotide polymorphism genotyping that mutations leading to a premature stop codon in inlA are common among Listeria monocytogenes isolates from ready-to-eat foods but not human listeriosis cases. 2010

Van Stelten, A., Simpson, J.M., Ward, T.J. and Nightingale, K.K.

Notes: Listeria monocytogenes uses the internalin A protein (InlA) to cross the intestinal barrier and cause foodborne illness. Mutations in inlA can introduce a premature stop codon, producing a truncated InlA protein that is secreted rather than associated with the bacterial cell wall. Strains with these inlA mutations have reduced virulence. The authors describe an inlA single nucleotide polymorphism (SNP)-based genotyping assay to distinguish isolates with the inlA mutations and use this assay to screen >1,000 L. monocytogenes isolates from ready-to-eat foods and human listeriosis cases. The assay involves amplification of the full-length inlA gene using GoTaq® Colorless Master Mix, purification of amplified products, then single-base-pair extension reactions. (4099)

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Cereb. Cortex 20, 2333–47. Serotonin 3A receptor subtype as an early and protracted marker of cortical interneuron subpopulations. 2010

Vucurovic, K., Gallopin, T., Ferezou, I., Rancillac, A., Chameau, P., van Hooft, J.A., Geoffroy, H., Monyer, H., Rossier, J. and Vitalis, T.

Notes: The authors characterized mouse neocortical interneurons that express 5-HT3A, a ligand-gated cation channel activated by 5-hydroxytryptamine (serotonin), during embryonic development. Transgenic mice that expressed green fluorescent protein (GFP) under the control of the 5-HT3A promoter were created. Single 5-HT3A-expressing neurons within 300µm brain sections of transgenic mice at various stages of embryonic development were subjected to whole-cell path-clamp recordings to examine their electrophysiological properties. To confirm activation of the 5-HT3A promoter in these cells, GFP expression was visualized by fluorescence microscopy without breaking the patch clamp seal. The contents of these single neurons then were aspirated and expelled into a 10µl reverse transcription reaction. After the reverse transcription, PCR was performed to simultaneously detect mRNAs encoding two isoforms of glutamic acid decarboxylase, three calcium-binding proteins, three neuropeptides, two transcription factors and reelin, a protein thought to be involved in neuronal migration and morphology. Two rounds of PCR using nested primers were required to detect these mRNAs. PCRs were performed using GoTaq® DNA Polymerase. Amplified products were visualized by agarose gel electrophoresis, using the 100bp DNA Ladder as a size standard. (4096)

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Development 137, 901–11. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. 2010

Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., Bösl, M.R., Hess, A., Surmann-Schmitt, C., von der Mark, H., de Crombrugghe, B. and von der Mark, K.

Notes: To study the role of the transcription factor Sox9 in the transition from cartilage to bone in newborn mice, the authors performed chromatin immunoprecipitation using the HaloCHIP™ System. Primary rib chondrocytes were transfected with a vector expressing full-length Sox9 with a HaloTag® protein tag, then proteins and DNA were cross-linked. DNA was isolated and sonicated, and the Sox9:DNA complexes were precipitated using the HaloLink™ Resin. The precipitated DNA then was amplified by PCR to determine that SOX9 binds to the SRY sites in the Vegfa gene. (4054)

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Nucl. Acids Res. 38, 6985-96. Targeted next-generation sequencing of DNA regions proximal to a conserved CXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer 2010

Chmielecki,J., Peifer, M., Socci, N.D., Hutchinson, K., Viale, A., Zhao, Z., Thomas, R.K. and Pao, W.

Notes: Human Genomic DNA:Male was used as a negative control in standard PCR and Sanger Sequencing to confirm fusion genomic breakpoints identified by NGS experiments. (4534)

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J. Am. Soc. Hort. Sci. 135, 291–302. Transcriptional profiling of rapidly growing cucumber fruit by 454-pyrosequencing analysis 2010

Ando, K. and Grumet, R.

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products prior to pyrosequencing. (4546)

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Genetics 182, 133–144. A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication. 2009

Blachon, S., Cai, X., Roberts, K.A., Yang, K., Polyanovsky, A., Church, A. and Avidor-Reiss, T.

Notes: These authors studied the formation of centrioles in Drosophila spermatids. Genomic DNA was extracted from whole flies using the Wizard® SV Genomic DNA Purification System. The DNA was then subjected to PCR, purified and sequenced. RNA was purified from whole flies using the SV Total RNA Isolation System. After RNA extraction, the samples were used in RT-PCR. (4064)

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J. Exp. Bot. 60, 1409-25. A strong effect of growth medium and organ type on the identification of QTLs for phytate and mineral concentrations in three Arabidopsis thaliana RIL populations. 2009

Ghandilyan, A., Ilk, N., Hanhart, C., Mbengue, M., Barboza, L., Schat, H., Koornneef, M., El-Lithy, M., Vreugdenhil, D., Reymond, M. and Aarts, M.G.

Notes: Mineral accumulation was studied in Arabidopsis thaliana comparing loci involved with growing in soil versus hydroponics. An F2 population derived from a cross between Landsberg erecta (Ler; maternal parent) and Eringsboda-1 (Eri-1; paternal parent) was propagated by single seed descent for nine successive generations in soil.
The flower buds of three plants per line were collected, and the DNA extracted using the Wizard® Magnetic 96 DNA Plant System and used for genotyping with 90 amplified fragment length polymorphism PCR (AFLP) and 39 single sequence length polymorphisms (SSLP) markers to build a genetic map of quantitative trait loci (QTL). (4136)

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J. Biol. Chem. 284, 26340–26348. Brain-derived neurotrophic factor enhances the basal rate of protein synthesis by increasing active eukaryotic elongation factor 2 levels and promoting translation elongation in cortical neurons. 2009

Takei, N., Kawamura, M., Ishizuka, Y., Kakiya, N., Inamura, N., Namba, H. and Nawa, H.

Notes: The authors studied how the basal rate of protein synthesis in primary cortical neurons was affected by chronic treatment of with a variety of neurotrophic factors and cytokines. Rat eukaryotic elongation factor 2 (eEF2) was cloned by PCR then subcloned into the pCI Mammalian Expression Vector and electroporated into neurons. After 72 hours, the neurons were harvested and used in various translation assays including ribosomal transit time. (4070)

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Appl. Environ. Microbiol. 75, 2275–83. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by gacA. 2009

Shi, X.Y., Dumenyo, C.K., Hernandez-Martinez, R., Azad, H. and Cooksey, D.A.

Notes: To gain a better understanding of how Xylella fastidiosa causes diseases in grapes, the authors mutated conserved regulatory genes, including gacA, that affect expression of virulence-related factors in other species. The relative expression levels of gacA in wildtype and mutated strains were examined using RT-PCR. The authors also identified and quantified a number of genes that were regulated by GacA by microarray analysis. Microarray results were confirmed using RT-PCR. RT-PCR was performed using the AccessQuick™ RT-PCR System. (4052)

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Drug Metab. Dispos. 37, 1726–1732. Characterizing the effects of common UDP glucuronosyltransferase (UGT) 1A6 and UGT1A1 polymorphisms on cis- and trans-resveratrol glucuronidation. 2009

Iwuchukwu, O.F., Ajetunmobi, J., Ung, D. and Nagar, S.

Notes: This study examined the genotype-phenotype correlation of the two major UGT isoforms, UGT1A1 and UGT1A6, involved in resveratrol metabolism. Genomic DNA was isolated from 30mg human liver tissue samples (normal and metastatic) using the Wizard® SV Genomic DNA Purification System. The purified DNA was eluted with 65°C water and 200–400ng of eluted DNA was used in a PCR-RFLP UGT1A6 genotyping assay. Amplification was carried out using PCR Master Mix in a final volume of 50µl, and the amplimers digested with appropriate restriction enzymes. (4018)

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Clin. Chem. 55, 748–56. Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR. 2009

Li, J., Wang, L., Jänne, P.A. and Makrigiorgos, GM.

Notes: The authors describe a new form of PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), to detect low-level somatic mutations. This technique is based on the facts that a) each DNA sequence has a critical denaturation temperature (Tc), which is lower than the melting temperature (Tm) and below which PCR efficiency decreases dramatically and b) Tc depends on DNA sequence. The authors used GoTaq® Flexi DNA Polymerase and mutation-specific TaqMan® probes for tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) to detect low-level somatic mutations in a mixture of wildtype and mutant DNAs. Conventional TaqMan® technology can detect mutant alleles at an abundance of 10–20% of that of the wildtype allele; using COLD-PCR the authors were able to increase selectivity 15- to 30-fold, detecting as little as 0.8% mutuant alleles. (4038)

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Nucl. Acids Res. 37, 7468–7482. Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides. 2009

Bertoni, C., Rustagi, A. and Rando, T.A.

Notes: This paper explored the effect of methyl-CpGmodified single-stranded oligonucleotides (ssODN) on gene repair. The Wizard® Genomic DNA Purification Kit was used to isolate gDNA from methylCpG-ssODN-treated myoblasts derived from limb muscle of neonatal C57 mice that stably expressed the mismatch repair site. One microgram of purified gDNA was digested overnight with 5U of restriction enzyme, purified, resuspended in 20µl of water and 5µl used in real-time PCR to determine if the target had been repaired. (4062)

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Arterioscler. Thromb. Vasc. Biol. Sept 24, [Epub ahead of print]. Eotaxin increases monolayer permeability of human coronary artery endothelial cells. 2009

Jamaluddin, M.S., Wang, X., Wang, H., Rafael, C., Yao, Q. and Chen, C.

Notes: Glutathione levels were assessed in human coronary artery endothelial cells (HCAECs) as a measure of oxidative stress. HCAECs were treated with either 100ng/ml eotaxin, a newly discovered chemokine, or pretreated with 2µmol/l MnTBAP for 30 minutes followed by eotaxin treatment for 45 minutes. Positive controls were treatment with 10µg/ml antimycin A and 2ng/ml TNF-α. Cellular glutathione was measured using the GSH-Glo™ Glutathione Assay. (4011)

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