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Methods in Mol. Biol. 577, 25-39. High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins 2009

Yamakawa, Hisashi

Notes: The authors use the Flexi® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag® Technology. They also describe a method of transferring ORFs between Flexi® Vectors in a 96-well plate format. They also used Wizard® SV 96 Plasmid DNA Purification, Wizard® SV PCR Clean-Up, and Wizard® SV Gel and PCR Clean-Up Systems. (4056)

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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. Clin. Endocrinol. Metab. 94, 2594-2601. KLF15 Is a transcriptional regulator of the human 17beta-hydroxysteroid dehydrogenase type 5 gene. A potential link between regulation of testosterone production and fat stores in women 2009

Du, X., Rosenfield, R. and Qin, K.

Notes: The authors used a HaloTag® vector and the HaloCHIP™ System to identify a KLF15 binding site in the HSD17B5 promoter. (4059)

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Vet. Rec. 164, 44–47. Prevalence of thermophilic Campylobacter species in household cats and dogs in Ireland. 2009

Acke, E., McGill, K., Golden, O., Jones, B.R., Fanning, S. and Whyte, P.

Notes: To examine the prevalence of Campylobacter species in asymtomatic carriers that can pass the bacteria onto humans, rectal swabs were collected from 147 dogs and 35 cats in Ireland and cultured on various diagnostic plates. The Wizard® Genomic DNA Purification System was used to isolate DNA from any suspect Campylobacter cultures. The purified DNA was used in multiplex PCR and RFLP to determine which species of Campylobacter was present. (4016)

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Proc. Natl. Acad. Sci. USA 106, 21306–11. Human cancers converge at the HIF-2alpha oncogenic axis. 2009

Franovic, A., Holterman, C.E., Payette, J. and Lee, S.

Notes: These authors used short heteronuclear RNAs (shRNA) in multiple cancer cell lines to silence hypoxia-inducing factor-2α (HIF-2α), a gene that many cancers exploit to increase angiogenesis and activate fundamental receptor tyrosine kinase signaling pathways to gain growth signal autonomy. Western blotting and RT-PCR were used to monitor the level of HIF-2α silencing. RT-PCR was performed using the AccessQuick™ RT-PCR System. The authors also examined the level of tyrosine kinase activation in shRNA-treated cells by Western blot analysis. To examine levels of ERK1/2 phopshorylation, the authors used the Anti-ERK 1/2 pAb to compare levels of activated and unactivated ERK1 and 2. (4050)

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Drug Metab. Dispos. 37, 1759–1768. Quantitative analysis of UDP-glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers. 2009

Izukawa, T., Nakajima, M., Fujiwara, R., Yamanaka, H., Fukami, T., Takamiya, M., Aoki, Y., Ikushiro, S., Sakaki, T. and Yokoi, T.

Notes: This study examined the expression levels of each UGT isoform in human liver and evaluated the variability between individuals. Total RNA from appropriate human tissues or various cell lines was used for RT-PCR of various human UDP-glucuronosyltransferases (UGT) cDNAs. The amplimers were cloned into the pTARGET™ Mammalian Expression Vector and verified by sequencing. The UGT vectors were linearized by restriction enzyme digestion and used for standards in real-time RT-PCR analysis. (4034)

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J. Biol. Chem. 284, 29526–35. Escherichia coli unsaturated fatty acid synthesis: complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB. 2009

Feng, Y. and Cronan, J.E.

Notes: The authors examined the role of two promoters in the regulation of fabA, an enzyme involved in unsaturated fatty acid synthesis. fabA transcript levels were quantified using real-time quantitative RT-PCR using ImProm-II™ Reverse Transcriptase, followed by a SYBR® Green method. (4053)

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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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Ann. Oncol. 20, 879-884. The importance of KRAS mutations and EGF61A>G polymorphism to the effect of cetuximab and irinotecan in metastatic colorectal cancer. 2009

Garm Spindler, K.L., Pallisgaard ,N., Rasmussen, A.A., Lindebjerg, J., Andersen, R.F., Crüger, D., and Jakobsen, A.

Notes: These authors used the Maxwell® 16 System to isolate genomic DNA from whole blood and normal colonic tissue samples. The DNA was used in genotype analysis, testing for wildtype and mutant KRAS genes, and for various EGFR-related polymorphisms. The results were used in a research study testing the relationship between various genotypes and response to different treatment regimens. (3961)

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Infect. Immun. 77, 3234–43. Sab, a novel autotransporter of locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21, contributes to adherence and biofilm formation. 2009

Herold, S., Paton, J.C. and Paton, A.W.

Notes: To try to understand the mechanism by which certain strains of Shiga-toxigenic E.coli adhere to host intestinal epithelium, the authors characterized the novel autotransporter protein Sab. Expression levels and protein localization were examined by Western blot analysis and enzyme-linked immunosorbent assay. The mouse anti-Sab antibody used in these studies was raised against an N-terminal His6-Sab fusion protein purified using the HisLink™ Resin. (4102)

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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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Nucl. Acids Res. 37, 5343–5352. Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance. 2009

Oberto, J., Breuil, N., Hecker, A., Farina, F., Brochier-Armanet, C., Culetto, E. and Forterre, P.

Notes: In this paper, the role of the KAE1/osgep/ygjD gene family, a universally conserved gene set without an assigned function, was investigated in yeast and Caenorhabditis elegans. Genomic DNA was isolated from Saccharomyces cerevisiae using the Wizard® SV Genomic DNA Purification System. This purified DNA was then used in PCR. (4065)

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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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J. Biol. Chem. 284, 3682–3690. Human flap endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance. 2009

Sampathi, S., Bhusari, A., Shen, B. and Chai, W.

Notes: The authors explored the role of a DNA replication factor, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. PCR was used to add a myc tag to the N terminus of FEN1 cDNA. The amplimer was gel purified, digested with NheI and SmaI, and cloned into the same sites in the pCI-neo Mammalian Expression Vector. The insert was confirmed by sequencing. (4030)

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Appl. Environ. Microbiol. 75, 5600–6. Single nucleotide polymorphism-based diagnostic system for crop-associated Sclerotinia species. 2009

Andrew, M. and Kohn, L.M.

Notes: The authors developed a single nucleotide polymorphism (SNP)-based assay to distinguish four Sclerotinia species. The assay consisted of amplification of a 300bp intergenic spacer and portions of the calmodulin and ras genes, followed by Southern blot using species-specific, radiolabeled probes. Amplifications were performed using the GoTaq® Colorless Master Mix, 0.2µM of each primer and 10–20ng of template DNA. (4098)

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J. Gen. Virol. 90, 1461–1470. Murid herpesvirus-4 lacking thymidine kinase reveals route-dependent requirements for host colonization. 2009

Gill, M.B., Wright, D.E., Smith, C.M., May, J.S. and Stevenson, P.G.

Notes: The authors examined the role of thymidine kinase (TK) in establishing a herpesvirus infection via the upper respiratory tract. DNA was purified from ex vivo organs of female BALB/c mice infected with a murid herpesvirus-4 (MuHV-4) TK knockout using the Wizard® Genomic DNA Purification Kit. Real-time PCR was used with 50–80ng of purified DNA to determine viral load of the animals. (4015)

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Plant Physiol. 150, 1356–1367. Sucrose control of translation mediated by an upstream open reading frame-encoded peptide. 2009

Rahmani, F., Hummel, M., Schuurmans, J., Wiese-Klinkenberg, A., Smeekens, S. and Hanson, J.

Notes: The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER® Vector and amino acid substitutions were introduced using the Altered Sites® II in vitro Mutagenesis System. The pGEM®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase® Reporter Assay System. (4023)

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Food Control [epub ahead of print]. Evaluation of DNA extraction procedures for traceability of various tomato products. 2009

Turci, M., Sardaro, M.L.S., Visioli, G., Maestri, E., Marmiroli, M. and Marmiroli, N.

Notes: In this study, the authors wanted to examine the ability to trace the origin of tomato goods from fresh to processed. They tested several DNA extraction procedures for fresh tomato, tomato sauce, tomato puree, tomato pulp, whole peeled S. Marzano PDO (Protected Designation of Origin) tomato, whole peeled tomato, tomato concentrate and ‘‘Arrabbiata sauce”. Homogenized material (200mg) was extracted in three replicates using seven different methods including the Wizard® DNA Clean-Up System. The DNA extracted was then analyzed by agarose gel electrophoresis, quantified and tested in PCR using SSR loci. The authors concluded that the Wizard® DNA Clean-Up System was the most effective of the DNA extraction methods tested and yielded the greatest number of successful amplification reactions with lowest investment of personnel time and money. (4003)

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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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Proc. Natl. Acad. Sci. USA 106(23), 9262-7. Long-lived Indy and calorie restriction interact to extend life span. 2009

Wang, P.Y., Neretti, N., Whitaker, R., Hosier, S., Chang, C., Lu, D., Rogina, B., and Helfand, S.L.

Notes: These authors investigated the relationship between calorie restriction and INDY expression on lifespan in Drosophila melanogaster. They showed that calorie restriction downregulates INDY expression in normal flies. INDY mutants on a normal diet had increased lifespan that was not extended further by calorie restriction. The INDY long-lived flies also shared several phenotypic characteristics with normal flies on a calorie-restricted diet. The authors then demonstrated that the phenotypic effects of the INDY mutation were not caused by reduced ability to intake food. The results show that INDY and calorie restriction interact to extend lifespan, and that decreased INDY expression induces a calorie-restriction-like status. During the study, the Maxwell 16 Tissue DNA Purification System was used to isolate DNA from Drosophila. The DNA was used in PCR analyses to confirm the absence of Wolbachia DNA in the experimental lines. (4001)

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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. Nutr. 139, 1054–1060. Folic acid supplementation during the juvenile-pubertal period in rats modifies the phenotype and epigenotype induced by prenatal nutrition. 2009

Burdge, G.C., Lillycrop, K.A., Phillips, E.S., Slater-Jefferies, J.L., Jackson, A.A. and Hanson, M.A.

Notes: This study examined the effects of folic acid supplementation on the offspring of pregnant rats fed a protein-restricted diet. Genomic DNA was extracted from rat adipose tissue using the Wizard® SV Genomic DNA Purification System. The purified DNA was then incubated with methylation-sensitive restriction enzymes and then used in real-time PCR. (4066)

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Clin. Can. Res. 15, 2523–2530. Identification of CD20 C-terminal deletion mutations associated with loss of CD20 expression in non-Hodgkin's lymphoma. 2009

Terui, Y., Mishima, Y., Sugimura, N., Kojima, K., Sakurai, T., Mishima, Y., Kuniyoshi, R., Taniyama, A., Yokoyama, M., Sakajiri, S., Takeuchi, K., Watanabe, C., Takahashi, S., Ito, Y. and Hatake, K.

Notes: The researchers examined if a CD20 mutation would affect resistance to rituximab, an adjunct cancer therapy drug used for CD20-positive B-cell lymphoma. CD20 PCR products amplified from genomic DNA were cloned into the pTARGET™ Mammalian Expression Vector. These CD20 mutant constructs were stably introduced into K562 chronic myelogenous leukemia cells by electroporation and selected using G-418. One microgram of CD20 mutant construct DNA was transcribed and translated using an in vitro translation kit from Promega. (4032)

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Nucl. Acids Res. 37, 2070–86. HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase IIα. 2009

Stros, M., Polanská, E., Struncová, S. and Pospísilová, S.

Notes: The authors examined whether HMGB1 and HMGB2 proteins could affect promoter activity of the topoisomerase IIα gene. Portions of the topoisomerase IIα gene promoter were cloned into the pGL3 Basic Vector, and Saos-2 cells were cotransfected with the resulting constructs, an HMGB1- or HMGB2-expressing plasmid and the pRL-tk Vector as a control for normalization. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay. To determine whether HMGB1 and HMGB2 promoted binding of the transcription factor nuclear factor-Y (NF-Y) to the topoisomerase IIα promoter, the authors used a chromatin immunoprecipitation (ChIP) assay. Two populations of Saos-2 cells, one of which expressed HMGB1 or HMGB2 and one that had expression inhibited, were fixed with formaldehyde, then treated to shear chromatin. Immunoprecipitation was performed using an anti-NF-Y antibody, and the amount of DNA bound to the NF-Y was quantified by semi-quantitative PCR using GoTaq® Hot Start DNA Polymerase and Green GoTaq® Flexi Reaction Buffer. (4037)

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Forensic Sci. Int. Genet. 3, 14–21. Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA. 2008

Krenke, B.E., Nassif, N., Sprecher, C.J., Knox, C., Schwandt, M. and Storts, D.R.

Notes: The authors describe the developmental validation of the Plexor® HY System, a quantitative PCR assay that simultaneously quantifies total human and male DNA. Validation studies examined: (1) human specificity, (2) sensitivity, (3) quantitation of degraded DNA, (4) impact of inhibitors, (5) male/female mixture and Y-assay male specificity, (6) reproducibility and concordance and (7) population studies. (3969)

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