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Veterinary Microbiology 160(3-4), 463–467. Molecular detection of murine noroviruses in laboratory and wild mice. 2012

Farkas, T., Fey, B., Keller, G., Martella, V. and Egyed, L.

Notes: Mice RNA samples were converted to cDNA using an oligo-dT primer with the Reverse Transcription System, ethanol precipitated, vacuum dried and transferred to another lab. There they were reconstituted in 20μl of molecular biology grade water.

Detection of caliciviruses in the wild mice samples was attempted using generic calicivirus primers targeting sequences encoding conserved amino acid motifs in the RNA-dependent RNA polymerase (RdRp) region of ORF1. Two microliters of cDNA was used in 25μl PCR reactions using the GoTaq® Green Master Mix. Laboratory mouse RNA samples were tested only with MNV-specific primers in the AccessQuick™ RT-PCR System using 2μl RNA as template.

PCR products were cloned into pGEM-T® Vector and sequenced using M13 forward and reverse primers on an ABI PRISM® 3730 DNA Analyzer. (4330)

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J. Immunol. 188(4), 1896–1904. Plac8-dependent and inducible NO synthase-dependent mechanisms clear Chlamydia muridarum infections from the genital tract. 2012

Johnson, R.M., Kerr, M.S. and Slaven, J.E.

Notes: The authors previously showed that there are two independent mechanisms by which Chlamydia-specific CD4 T cells clear infection in epithelial cells; an iNOS-dependent mechanism and a Plac8-dependent mechanism. To further identify the Plac8 mechanism, they used microarrays to identify a second mechanism dependent on Plac8 for terminating Chlamydia replication in epithelial cells.

Several Chlamydia-specific CD4 T cell clones were purified at the end of their culture cycle and grown for 3 days in their usual culture media plus growth factors, without Ag stimulation. Total RNA was isolated from each clone using a protocol that included an RNase-free DNase I treatment step. Specific mRNA gene reverse transcription and amplification were performed using the AccessQuick™ RT-PCR System. (4324)

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J. Biomol. Tech. 23, 4-10. Random amplification and pyrosequencing for identification of novel viral genome sequences. 2012

Hang, J., Forsheym, B.M., Kochel, T.J., Li, T., Solórzano, V.F., Halsey, E.S., and Kuschner, R.A.

Notes: This paper describes a method for sequencing unknown viral isolates from tissue culture using anchored random reverse transcription and PCR, pyrosequencing and data analysis. RNA was extracted from tissue culture supernatants positive for viral antigens and used in RT-PCR with random primers. Amplification products were gel-purified and used in pyrosequencing reactions. A QuantiFluor™-P Fluorometer was used to measure copy number concentration relative to a standard, prior to Roche 454 pyrosequencing. (4231)

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Appl. Environ. Microbiol. 78, 445–54. Responses of methanogen mcrA genes and their transcripts to an alternate dry/wet cycle of paddy field soil. 2012

Ma, K., Conrad, R. and Lu, Y.

Notes: The authors of this study investigated the microbial mechanisms associated with the reduction of methane production and emission from rice fields observed with intermittent field drainage. They looked in particular at the abundance of mcrA gene copies and transcripts from rice paddy soil fauna. The mcrA gene encodes the alpha subunit of methyl coenzyme M reductase. 

Total nucleic acid was extracted from soil samples using a phenol-chloroform procedure. For RNA analyses, DNA was hydrolyzed using RQ1 RNase-free DNase in the presence of 0.2µl Recombinant RNasin® Ribonuclease Inhibitor and then further purified using a commercial kit. cDNA synthesis was carried out using the Improm-II™ Reverse Transcription System, again in the presence of 1.0µl Recombinant RNasin® Ribonuclease Inhibitor. A clone library of transcripts was generated using the pGEM®-T Easy Vector System. The transcript standard for quantitative mcrA analysis was prepared from the in vitro transcript of a mcrA clone using the Riboprobe® in vitro Transcription Systems. (4241)

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J. Biomed. Sci. 19, 42. Shikonin enhances efficacy of a gene-based cancer vaccine via induction of RANTES. 2012

Chen, H.M., Wang, P.H., Aravindaram, K., Chen, Y.H., Yu, H.H., Yang, W.C., and Yang, N.S.

Notes: In this study, the authors evaluated whether application of the phytochemical shikonin to the skin of mice was able to augment the effect of a DNA-based anti-tumor vaccine by inducing the cytokine RANTES. As part of the study, the AccessQuick™ System was used in RT-PCR analysis to determine expression of RANTES mRNA in treated and control skin samples. (4288)

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J. Biol. Chem. 286, 37196–206. 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-myc nuclear localization and binding to the e-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes. 2011

Ning, B., Liu., G., Liu, Y., Su, X., Anderson, G.J., Zheng, X., Chang, Y., Guo, M., Liu, Y., Zhao, Y. and Nie, G.

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

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J. Clin. Microbiol. 49, 281–291. Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers. 2011

Stressmann, F.A., Rogers, G.B., Klem, E.R., Lilley, A.K., Donaldson, S.H., Daniels, T.W., Carroll, M.P., Patel, N., Forbes, B., Boucher, R.C., Wolfgang, M.C. and Bruce, K.D.

Notes: Sputum samples were collected from cystic fibrosis patients and 16S rRNA sequences amplified by PCR. These products were cloned into a T-vector, transformed into competent cells and the resulting colonies grown in 2ml LB broth in 96-deep-well plate for 20 hours. Of this culture, 1.9ml was pelleted and the clones isolated using the Wizard® SV Plasmid Purification System. The purified plasmid DNA was subjected to agarose gel electrophoresis and sequenced. (4133)

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Acta Pharmacologica Sinica 32, 368-74. Attenuated Salmonella typhimurium carrying shRNA-expressing vectors elicit RNA interference in murine bladder tumors. 2011

Yang, N., Li, S.H., Lü, Y.Z., Chen, L.S., and Ren, D.M.

Notes: This proof-of-principle study investigated whether attenuated Salmonella typhimurium could be used as a vehicle for delivering shRNA-expressing plasmid DNA into cancer cells in mice. The authors delivered S. typhimurium bearing plasmids encoding anti-GFP shRNA orally to mice harboring tumors that espressed GFP. They were able to show that the bacteria accumulated and persisted for 40 days within the tumors, and that GFP expression in infected tumors was reduced. The AccessQuick™ RT-PCR System was used to analyze GFP expression levels in cultured cells and tumors. (4347)

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Biochem. Biophys. Res. Commun. 408, 160-166. Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells. 2011

 Yu, K.S., Jo, J.Y., Kim, S.J., Lee, Y., Bae, J.H., Chung, Y.H., and Koh, S.S.

Notes: These authors showed that expression of the serine protease inhibitor elafin is regulated by epigenetically controlled expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, resulting in reduced proliferation and increased apoptosis. Luciferase reporter assays were used to show that Foxa2 binding was required for activation of elafin expression, and that Foxa2 binding was activated by treatment with the methyltransferase inhibitor. These assays used a pGL3-Basic Vector construct in which expression of firefly luciferase was driven by the upstream region bearing the Foxa2 binding site. The pRL-TK Vector, expressing Renilla luciferase, was used as a normalization control. The AccessQuick™ System was used for RT-PCR analysis to show that Foxa2 mRNA was barely detectable in melanoma cells.  (4345)

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Cytokine 55, 79-89. GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells. 2011

Wei, W.C., Su, Y.H., Chen, S.S., Sheu, J.H., and Yang, N.S.

Notes: This study compared the effects of zymosan and LPS on human monocyte-derived dendritic cells (DCs). The  authors found that zymosan-activated DCs had a unigue cytokine expression profile. In zymosan-activated DCs, high levels of GM-CSF and IL-27, rather than IL-12 p70, were involved in Th1 cell activation. As part of the study, RT-PCR was used to investigate the molecular basis of this failure to induce production of active IL-12 p70. Expression levels of the biologically inactive subunits of IL-12 p70 (p40 and p35) was assessed using the AccessQuick™ RT-PCR System. The results showed that zymosan induced expression of p40, but not p35 mRNA, indicating that lack of induction of p35 was the reason for failure to induce active IL-12 p70. (4348)

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PLos ONE 6(9), e24417. Metagenome plasticity of the bovine abomasal microbiota in immune animals in response to Ostertagia ostertagi infection. 2011

Li, R.W., Wu, S., Li, W., Huang, Y., and Gasbarre, L.C.

Notes: These authors performed metagenomic analysis to investigate any changes in composition of the microbial flora of the abomasa (ruminant 4th stomach compartment) in immune cattle after reinfection with the nematode Ostertagia ostertagi. DNA was extracted from abomasal contents using a QIAamp stool kit and the integrity verified using an Agilent Bioanalyzer 2000. PCR was then performed using primers targeting hypervariable regions of 16S rRNA genes. The amplified material was gel-purified and sequenced using the Roche 454 pyrosequencing system. DNA concentration was measured before and after PCR using the QuantiFluor™ Fluorometer.



(4230)

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Infect. Immun. 79, 2451-9. The Agr-like quorum-sensing system regulates sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A non-food-borne human gastrointestinal disease strain F5603. 2011

Li, J., Chen, J., Vidal, J.E., and  McClane, B.A.

Notes: This study explored whether the Agr-like quorum-sensing system regulates sporulation and production of the Clostridium perfringens toxins CPE and beta2 toxin. An agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, had reduced production of alpha-toxin and perfringolysin O during vegetative growth, and did not form spores efficiently. The AccessQuick™ System was used in RT-PCR analysis to confirm the presence or absence of agrB transcripts in the wild type, mutant, and complemented strains used in the study. (4546)

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Nucl. Acids Res. 39, e81. A method for counting PCR template molecules with application to next-generation sequencing. 2011

Casbon, J.A., Osborne, R.J., Brenner, S. and Lichtenstein, C.P.

Notes: DNA templates are often amplified by PCR during library generation prior to next-generation sequencing, but amplification can introduce biases and duplications that are not easily corrected. In this paper, the authors developed a simple method to count the number of input template molecules to reduce these PCR-related problems: The ligation of a degenerate base region to all fragments during library creation. To evaluate their approach to correct for biases and duplications, the authors created a library using Human Genomic DNA, amplified the library by inverse PCR using the GoTaq® Hot Start Polymerase and 1X Colorless GoTaq® Flexi Buffer, sequenced the resulting DNA fragments and assessed the quality of the next-generation sequencing data. (4160)

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Hum. Mol. Genet. 21, 577–85. A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing 2011

Soldà, G., Robusto, M., Primignani, P., Castorina, P., Benzoni, E., Cesarani, A., Ambrosetti, U., Asselta, R. and Duga, S.

Notes: To confirm the role of a mutation in the miR-96 microRNA (miRNA) associated with an autosomal dominant hearing lost, HeLa cells (250,000 cells per well in six-well plates) were transfected with 4µg of plasmid carrying wild type or mutant miR-96 miRNA using FuGENE® HD Transfection Reagent. After 24 hours, the cells were washed and total RNA extracted. After quantitation, the RNA used in RT-PCR analysis. The entire 3´UTRs of eight putative target genes were amplified by PCR from genomic DNA and cloned into the psiCHECK™-2 Vector. HeLa cells were transiently transfected with 2µg of the 3´ UTR psiCHECK™-2 constructs and 0.2µg of a wild-type, single or double mutant miR-96 plasmid using FuGENE® HD Transfection Reagent. Forty-eight hours after transfection, the Dual-Luciferase® Reporter Assay System was used to quantitity the firefly and Renilla luciferase in cell lysates. (4251)

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J. Biol. Chem. 286, 42690-42703. Alternative Splicing Produces Nanog Protein Variants with Different Capacities for Self-renewal and Pluripotency in Embryonic Stem Cells. 2011

Das, S., Jena, S., and Levasseur, D.N.

Notes: The transcription factor Nanog is required for the maintenance of embryonic stem (ES) cell pluripotency. These authors showed that the Nanog N-terminal domain is regulated by post-transcriptional modification, and that alternative splicing generates Nanog variants with different capacities for maintaining an undifferentiated cell state. As part of their study, the authors used GoScript® Reverse Transcriptase to to generate cDNA from RNA extracted from cell lines expressing different Nanog variants. The cDNA was used in RT-qPCR to quantify relative expression levels.
(4184)

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Blood Cells Mol. Dis. 46, 139-144. Application of MLPA assay to characterize unsolved α-globin gene rearrangements. 2011

Colosimo, A,. Gatta, V., Guida, V., Leodori, E., Foglietta, E., Rinaldi, S., Cappabianca, M.P., Amato, A., Stuppia, L., and Dallapiccola, B.

Notes: These authors used the Maxwell® 16 Blood DNA Purification Kit to isolate genomic DNA from leukocytes. (4210)

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J. Microbiol. Methods 81, 127-134. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. 2011

Salonen, A., Nikkilä, J., Jalanka-Tuovinen, J., Immonen, O., Rajilić-Stojanović, M., Kekkonen, R.A., Palva, A., and de Vos, W.M.

Notes: These authors compared the performance of four DNA purification methods for recovery of bacterial and archaeal DNA from fecal material. One of the methods tested was the Wizard® Genomic DNA Purification Kit, which uses a solution-based, enzymatic method for extraction. The Wizard® Genomic method was rated highly on extraction speed, and gave the highest DNA yields. A second method involving mechanical disruption (repeated bead beating) was rated more highly on extraction efficiency from archaea and some bacterial species. The criteria for performance comparison are described fully in the paper. (4219)

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Appl. Environ. Microbiol. 77, 2589–95. Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification. 2011

Han, F., Wang, F. and Ge, B.

Notes: The authors developed a loop-mediated isothermal amplification (LAMP) assay to distinguish between virulent and nonvirulent strains of Vibrio vulnificus by targeting the virulence-correlated gene (vcg). The authors performed PCR using vcg-specific primers and GoTaq® Hot Start Polymerase in parallel to confirm the LAMP results. (4163)

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J. Clin. Microbiol. 49(1), 335-44. Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.
2011

Medina, R.A., et al.

Notes: These authors used extensive computational analysis of isolates from the 2009 H1N1 outbreak to identify conserved H1N1 sequence signatures that could potentially be used in diagnostic assays to track the spread of specific strains during viral outbreaks. They identified target sequences in the hemagglutinin and neuraminidase genes that were highly conserved among 2009 H1N1 isolates. They then designed primers to amplify those sequences and used them in Taqman® and SYBR® Green-based qPCR assays to create a discriminatory 2009 H1N1 detection assay. They used the AccessQuick™ System in conventional RT-PCR to first establish whether their chosen primers were specific for the 2009 H1N1 isolates. In that assay they amplified representative H1N1 strains spanning from 1930 to the 2009 H1N1, and showed that only the 2009 isolates generated product. (4284)

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J. Antimicrob. Chemother. 67, 59-63. Disruption of the blaOXA-51-like gene by ISAba16 and activation of the blaOXA-58 gene leading to carbapenem resistance in Acinetobacter baumannii Ab244.
2011

Lopes. B.S., Evans, B.A., and Amyes, S.G.B.

Notes: This study investigated the genetic basis of carbapenem resistance in the multidrug resistant Acinetobacter baumannii isolate, Ab244. Transposable elements are known to play an important role in multidrug resistance in A. baumannii. The authors used a multiplex PCR approach to detect the presence of known resistance genes and insertion elements, followed by RT-PCR to study expression of the genes identified. For RT-PCR, cDNA was synthesized from 100 ng of RNA using the AccessQuick™ RT-PCR System. Results of the study indicated that the blaOXA-132 gene was inactivated in A. Baumannii AB244 by insertion of ISAba16, and that carbapenem resistance in that isolate was due to an alternate resistance mechanism caused by overexpression of the blaOXA-58 gene. (4344)

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Sci. Signal. 4, ra51. Distinct Phosphorylation Sites on the β2-Adrenergic Receptor Establish a Barcode That Encodes Differential Functions of β-Arrestin 2011

Nobles, K.N., Xiao, K., Ahn, S., Shukla, A.K., Lam, C.M., Rajagopal, S., Strachan, R.T., Huang, T.Y., Bressler, E.A., Hara, M.R., Shenoy, S.K., Gygi, S.P. and Lefkowitz, R.J.

Notes: The authors created a stably transfected HEK293 cell line expressing a luminogenic cAMP-binding protein using GloSensor™ technology to quantify cAMP levels in live cells. The HEK293 cells express a beta2-adrenergic receptor (β2AR ), a Gs-coupled receptor, that when activated with an agonist, stimulates the production of cAMP. The cell line was used to demonstrate that the phosphorylation pattern (“barcode”) of the β2AR created by various G protein-coupled receptor kinases (GPKs) affects the binding and function of beta-arrestin and subsequent internalization of β2AR. GloSensor™ transfection and transcription were confirmed by stimulation of endogenous β2AR with isoproterenol. Endogenous β2ARs in HEK293 cells were prestimulated with either vehicle DMSO or isoproterenol, then washed and rechallenged with serially diluted isoproterenol. In cells transfected with control siRNA, pretreatment with isoproterenol induced a 50% loss of the maximal cAMP signal when rechallenged. Cells transfected with siRNA targeting GRK2, GRK6, or both GRK2 and GRK6 showed impairment of this desensitization. This GRK knockdown effect decreased the change observed in the maximal cAMP response (Emax) after isoproterenol pretreatment. (4138)

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mBio. 2(6), e00275-11. Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system. 2011

Chen, J., Rood, J.I., and McClane, B.A.

Notes: These authors investigated whether ETX toxin production in C. perfringens type D is regulated by the Agr-like quorum sensing system, the VirS/VirR system, or both. They demonstrated that an agr mutant lacked ETX expression, and showed that lack of VirR had no effect on ETX production. The AccessQuick™ System was used in RT-PCR analysis of RNA isolated from mutant, wildtype and reconstituted (complemented) strains to confirm absence of agr transcripts in mutant strains. RT-PCR was also used to confirm the presence of etx transcripts in wild type strains, and their absence in the agr mutant. Previous studies had shown that toxin production is upregulated upon contact with enterocyte-like Caco-2 cells. This study showed that that the agr operon is required for such upregulation. (4289)

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Appl. Environ. Microbiol. 77, 2943–53. Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces. 2011

Kwan K., Cooper M., La Duc M.T., Vaishampayan P., Stam C., Benardini J.N., Scalzi G., Moissl-Eichinger C. and Venkateswaran K.

Notes: These authors used the Maxwell® 16 System to extract DNA from multiple sample collection devices containing a model microbial community (MMC) comprised of 11 distinct species of bacterial, archaeal and fungal lineages associated with spacecraft or clean-room surfaces. The authors compared cotton swabs, polyester wipes and biological sampling kits to assess the success of recovering DNA of rRNA genes for species-specific PCR analysis. (4124)

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Virol. J. 8, 387. First report of multiple lineages of dengue viruses type 1 in Rio de Janeiro, Brazil. 2011

dos Santos, F.B., Nogueira, F.B., Castro, M.G., Nunes, P.C., de Filippis, A.M., Fariam, N.R., Simões, J.B., Sampaio, S.A., Santos, C.R. and Nogueira, R.M.

Notes: The authors examined the strains of Dengue virus serotype 1 (DENV-1) found in the State of Rio de Janeiro since it was introduced in 1986. Viral RNA was extracted from patient serum samples or cell culture and 5µl of RNA reverse transcribed and amplified using the AccessQuick™ RT-PCR System with primers for the 2,325bp C/prM/M/E region of DENV-1. The products were sequenced and aligned to examine how the virus had evolved over time. (4342)

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Arch. Med. Sci. 7, 501-507. Frequency of Firmicutes and Bacteroidetes in gut microbiota in obese and normal weight Egyptian children and adults. 2011

Ismail, N.A., Ragab, S.H., Elbaky, A.A, Shoeib, A.R., Alhosary, Y., and Fekry, D.

Notes: These authors investigated the differences in gut microbial flora between obese and normal-weight subjects. They used the Wizard® Genomic DNA Purification Kit to extract DNA from diluted fecal extracts. The extracted DNA was analyzed by PCR to identify Bacteroidetes and Firmicutes. Differences in distribution of these phyla was between the subject groups were identified. (4220)

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