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Infect. Immun. 75, 3478–89. Reversal of the antichlamydial activity of putative type III secretion inhibitors by iron. 2007

Slepenkin, A., Enquist, P.A., Hägglund, U., de la Maza, L.M., Elofsson, M. and Peterson, E.M.

Notes: The authors screened members of a class of acylated hydrazones of salicylaldehydes (INPs) to characterize their ability to inhibit growth of Chlamydia by affecting the type III secretion (T3S) system, a potent virulence mechanism. Expression levels of various T3S genes and gene markers of early, middle and late developmental cycles were examined by RT-PCR in Chlamydia trachomatis-infected HeLa 229 cells in the presence and absence of INPs. HeLa229 RNA was isolated at 4, 8, 24 and 36 hours postinfection, treated with RQ1 RNase-Free DNase and amplified using the Access RT-PCR System in the presence of 0.5 units of RNasin RNase Inhibitor. Each set of experiments included a no-reverse transcriptase control reaction to control for DNA contamination and a positive control reaction using the Positive Control RNA with Carrier and control primers supplied with the kit. (3795)

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Exp. Biol. Med. 232, 1195–1203. 13-cis-Retinoic acid alters intracellular serotonin, increases 5-HT1A receptor, and serotonin reuptake transporter levels in vitro. 2007

O'Reilly, K.C., Trent, S., Bailey, S.J. and Lane, M.A.

Notes: The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers. (3790)

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Cancer Res. 67, 10600–10607. Interaction of the tumor metastasis suppressor nonmetastatic protein 23 homologue H1 and estrogen receptor alpha alters estrogen-responsive gene expression. 2007

Curtis, C.D., Likhite, V.S., McLeod, I.X., Yates, J.R. and Nardulli, A.M.

Notes: Tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1) interacts with estrogen receptor α (ERα) and influences ERα-mediated gene expression. The authors knocked down NM23-H1 expression using RNA interference in estrogen-treated or untreated MC-7 human breast cancer cells and determined the effect on transcription of estrogen-responsive genes, including progesterone receptor, Bcl-2, cathepsin D and cyclin D1. Levels of these mRNAs were measured in the presence of NM23-H1 or control small interfering RNAs using quantitative RT-PCR. Total RNA was treated with RQ1 RNase-Free DNase to remove contaminating DNA, and cDNA was synthesized using the Reverse Transcription System. The resulting cDNA was subjected to quantitative PCR using SYBR® Green dye. (3789)

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J. Biol. Chem. 282, 21818–21828. Degradation of hsp70 and other mRNAs in Drosophila via the 5´ 3´ pathway and its regulation by heat shock. 2007

Bönisch, C., Temme, C., Moritz, B. and Wahle, E.

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-32P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq® DNA Polymerase, 1.5mM MgCl2 and 1X Green GoTaq® Flexi Reaction Buffer. (3707)

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J. Forensic Sci. 52, 1073–6. Characterization of the variant allele 9.2 of Penta D locus. 2007

Miozzo, M.C., Maxzud, M.K., Pacharoni, C.M., Mutal, S.A. and Modesti, N.M.

Notes: During casework analysis using the PowerPlex® 16 System, the authors identified an off-ladder allele at the Penta D locus as the microvariant allele 9.2. DNA from individuals with the 9.2 allele was amplified using a single primer pair specific for Penta D, and the amplification products were purified and sequenced to characterize the microvariant allele. PCR products were purified using the Wizard® PCR Preps DNA Purification System. Sequence analysis revealed that the 9.2 allele has 10 STR repeats and a TAA deletion in the 3´ flanking region. (3771)

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J. Clin. Microbiol. 45, 1469–1477. Multilocus sequence typing of the pathogenic fungus Aspergillus fumigatus. 2007

Bain, J.M., Tavanti, A., Davidson, A.D., Jacobsen, M.D., Shaw, D., Gow, N.A. and Odds, F.C.

Notes: The authors developed a multilocus sequence typing scheme (MLST) to examine sequence variation and discriminate between Aspergillus fumigatus strains. They also examined the distribution of MAT1-1 and MAT1-2 sexual idiomorphs in 100 clinical and environmental isolates. Sexual idiomorphs were determined using PCR and a reverse primer to both idiomorphs and a forward primer specific to either MAT-1 or MAT-2. PCRs consisted of 2mM MgCl2, 200µM DNTPs and 2.5 units of GoTaq® DNA Polymerase. (3714)

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J. Biol. Chem. 282, 21798–21809. Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed. 2007

Panis, G., Méjean, V. and Ansaldi, M.

Notes: The authors studied the defective prophage KplE1 in E. coli K12 to map the binding sites of proteins required for recombination. Prior to in vivo excision assays in two E. coli K12 strains, the presence of three DNA sequences required for recombination was confirmed by PCR using GoTaq® DNA Polymerase. In vitro excision assays were also performed using linear and supercoiled DNA substrates that were purified using the Wizard® PCR Clean-Up System. Finally the phage-encoded integraseS (IntS) mRNA was quantitated by real-time RT-PCR. The RNA template was purified from E. coli K12 using the PureYield™ RNA Midiprep System. (3722)

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J. Mol. Endocrinol. 36, 449–461. Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo. 2007

Brown, K.A., Boerboom, D., Bouchard, N., Doré, M., Lussier, J.G. and Sirois, J.

Notes: The authors cloned the novel equine aldo-keto reductase AKR1C23 and characterized its expression patterns in the preovulatory follicle. The AKR1C23 cDNA was amplified from equine ovarian RNA using the Access RT-PCR System and primers designed by sequence alignments of known AKR sequences, then cloned into the pGEM®-T Easy Vector. Levels of AKR1C23 and ribosomal protein L17a mRNAs in various equine tissues were quantified using the Access RT-PCR System and 21 cycles and 18 cycles, respectively, followed by agarose gel electrophoresis, transfer to nylon membranes, and hybridization to radiolabeled probes synthesized using the Prime-a-Gene® Labeling System. (3791)

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J. Immunol. 179, 4829–4839. Aging up-regulates expression of inflammatory mediators in mouse adipose tissue. 2007

Wu, D., Ren, Z., Pae, M., Guo, W., Cui, X., Merrill, A.H. and Meydani, S.N.

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

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J. Clin. Invest. 117, 3042–3048. HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection. 2007

McAulay, K.A., Higgins, C.D., Macsween, K.F., Lake, A., Jarrett, R.F., Robertson, F.L., Williams, H. and Crawford, D.H.

Notes: The authors examined whether genetic differences at the HLA class I locus affect development of Epstein Barr Virus-associated diseases. Peripheral blood mononuclear cells were isolated from asymptomatic EBV-seropositive and seronegative individuals and patients with acute infectious mononucleosis. DNA was isolated, and genotypes at two HLA class I loci and one HLA class III locus, as a control, were determined by PCR. The 10µl PCRs contained 25ng of DNA, 1X GoTaq® Flexi Reaction Buffer, 2.5mM MgCl2, 200µM dNTP, 0.5 units of GoTaq® Flexi DNA Polymerase and 25µM of forward and reverse primer, one of which was labeled with 6-FAM fluorescent dye. The results show that HLA class I polymorphisms might predispose people to develop infectious mononucleosis upon EBV infection. (3712)

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Proc. Natl. Acad. Sci. USA 104, 10637–10642. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. 2007

Rendón, M.A., Saldaña, Z., Erdem, A.L., Monteiro-Neto, V., Vázquez, A., Kaper, J.B., Puente, J.L. and Girón, J.A.

Notes: The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector containing the ecpA gene, restored the strain's ability to adhere to epithelial cells. (3719)

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Appl. Environ. Microbiol. 74, 811-817. High frequency of histamine-producing bacteria in enological environment and instability of the phenotype. 2007

Lucas, P.M., Claisse, O. and Lonvaud-Funel, A.

Notes: Because of concerns about the consumption of histamine in wine (which makes histamine more potent), the quantity of lactic acid bacteria (LAB), which can produce histamine during winemaking, was determined in 264 samples of red wine from 116 wineries. DNA was isolated from LAB strains grown in De Man Rogosa Sharpe (MRS) broth using the Wizard® Genomic DNA Purification Kit. Glass beads were used to disrupt the cells, and a modified Wizard® Genomic DNA Purification protocol that included PVP treatment was performed. The purified DNA was used in qPCR analysis. (3741)

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Am. J. Pathol. 171, 1153-1167. Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy. 2007

Bodó, E., Tobin, D.J., Kamenisch, Y., Bíró, T., Berneburg, M., Funk, W. and Paus, R.

Notes: To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan® Assay. (3746)

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Assay Drug Dev. Technol. 5, 237–245. A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP 2007

Kumar, M., Hsiao, K., Vidugiriene, J. and Goueli, S.A.

Notes: The authors of this paper introduce a luminescent assay to monitor changes in cellular cAMP concentration. The assay can be used to study the activity of G-protein coupled receptors that modulate adenylate cyclase activity. The assay is compatible with high-throughput screening in 96-, 384- and 1536-well formats. (3928)

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J. Biol. Chem. 282, 29211–21. A novel CaV1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits. 2007

Cheng, X., Liu, J., Asuncion-Chin, M., Blaskova, E., Bannister, J.P., Dopico, A.M. and Jaggar, J.H.

Notes: The authors identified a novel subunit of the voltage-dependent L-type Ca2+ channel (CaV1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM®-T Easy Vector and sequenced using the T7 Promoter Primer. (3801)

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Mol. Cell. Biol. 26, 8448–8460. Specific isoforms of translation initiation factor 4GI show differences in translational activity. 2007

Coldwell, M.J. and Morley, S.J.

Notes: The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function. (3778)

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Genetics 175, 1047-1058. Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes. 2007

Lamb, J.C., Danilova, T., Bauer, M.J., Meyer, J.M., Holland, J.J., Jensen, M.D., and Birchler, J.A.

Notes: These authors generated a set of probes that could be used in fluorescence in situ hybridization (FISH) analyses for karyotyping studies on maize chromosomes. Specific target regions composed of genes or gene clusters and free from repetetive elements were identified for each chromosome. Target regions were amplified by PCR, gel purified using the Wizard® SV Gel and PCR Clean-Up System, and tested in a FISH assay. Probes showing low background were selected, subcloned into the pGEM® -T Vector and sequenced to confirm identity. (3627)

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Appl. Environ. Microbiol. 73, 2860-2870. The microbial community structure in petroleum-contaminated sediments corresponds to geophysical signatures. 2007

Allen, J.P., Atekwana, E.A., Atekwana, E.A., Duris, J.W., Werkema, D.D., and Rossbach, S.

Notes: These authors studied microbial community structure at various locations in an aged underground petroleum plume. DNA was purified from soil samples collected from different sites within a contaminated area. 16S rRNA genes were then amplified from the isolated DNA, and the PCR products were run on a gel and purified using the Wizard® SV Gel and PCR Clean-Up System. After subcloning into a TA vector, the 16S RNA genes were sequenced and used to identify the various Phyla represented and characterize the microbial populations present throughout the site. (3625)

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J. Virol. Methods 144, 86–90. A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus. 2007

Prichard, M.N., Daily, S.L., Jefferson, G.M., Perry, A.L. and Kern, E.R.

Notes: The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3761)

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Mol. Cancer Ther. 6, 856-865. The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and has antitumor activity against gastrointestinal adenocarcinoma tumor xenografts in vivo. 2007

Loo, D., Pryer, N., Young, P., Liang, T., Coberly, S., King, K.L., Kang, K., Roberts, P., Tsao, M., Xu, X., Potts, B. and Mather, J.P.

Notes: The authors examined the effectiveness of a monoclonal antibody treatment to human tumor-derived cells implanted under the kidney capsule of male athymic mice. These tumors were recovered from mouse kidney and the total genomic DNA isolated using the Wizard® SV Genomic DNA Purification System. The human tumor DNA was quantified using a TaqMan® qPCR method. (3748)

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J. Biol. Chem. 282, 14364–14372. Expression of sialidase Neu2 in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinases signaling. 2007

Tringali, C., Lupo, B., Anastasia, L., Papini, N., Monti, E., Bresciani, R., Tettamanti, G. and Venerando, B.

Notes: The authors transfected the myleoid leukemic cell line K562 with the cytosolic sialidase Neu2. Expression of Neu2 resulted in a significant decrease in mRNA levels for the anti-apoptotic factors Bcl-XL and Bcl-2 as determined by real-time PCR. Reverse transcription was carried out with 1µg of total RNA using the ImProm-II™ Reverse Transcription System and random hexamers. cDNA representing 10ng of total RNA was used in a real-time PCR to quantitate Bcl-XL and Bcl-2 mRNA levels. (3725)

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J. Biol. Chem. 282, 14194-14204. Regulation of the interleukin-7 receptor α-promoter by the Ets transcription factors PU.1 and GA-binding protein in developing B cells. 2007

Dekoter, R.P., Schweitzer, B.L., Kamath, M.B., Jones, D., Tagoh, H., Bonifer, C., Hildeman, D.A., and Huang, K.J.

Notes: The interleukin-7 receptor is composed of γ and α subunits, encoded by the genes il7rg and il7r, respectively. The α subunit is expressed in developing B cells and is downregulated upon maturation. These authors investigated the mechanisms of transcriptional regulation of the il7r gene using 5´ RACE, EMSA, RNA interference and chromatin immunoprecipitation analyses. Potential promoter regions identified by 5´ RACE analysis were cloned into the pGL3-Basic luciferase reporter vector for further study. The promoter constructs were transiently transfected into the 38B9 pro-B cell line along with the control pRL-TK Vector, which expresses Renilla luciferase, and the Dual-Luciferase® Reporter Assay System was used to assess luciferase activity from the various promoter constructs. The promoter construct having the highest activity was chosen, and site directed mutagenesis was used to identify specific regions within the promoter fragment that may be important for activity. Sequence analysis was then used to identify a conserved Ets transcription factor binding site within the putative il7r promoter region. To determine whether the ETS transcription factor GABP binds to this Ets region, the authors performed chromatin immunoprecipitation analysis with an anti-GABP antibody. Immunoprecipitated DNA was then PCR-amplified with primers specific for the Ets region or control primers. The Wizard® SV Gel and PCR Clean-Up System was used to purify the amplified fragments prior to semiquantitative PCR analysis. (3626)

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J. Virol. 81, 173–81. In vivo packaging of brome mosaic virus RNA3, but not RNAs 1 and 2, is dependent on a cis-acting 3' tRNA-like structure. 2007

Annamalai, P. and Rao, A.L.

Notes: The brome mosaic virus (BMV) contains four RNAs (B1, B2, B3 and B4), each of which includes a tRNA-like structure (TLS) that is required for packaging in vitro. To confirm the necessity of the TLS for packaging in vivo, the authors created TLS-less B1, B2 and B3 RNAs; TLS-less B1 and B2 RNAs were packaged, while the TLS-less B3 RNA was not. Co-infiltration of Nicotiana benthamiana leaves with wildtype B1 and B2 and TLS-less B3 RNA resulted in restoration of the B3 TLS. To determine whether the resulting TLS was regained by homologous or heterologous recombination with B1 or B2 RNA, the 3´ end of the B3 RNA was amplified from leaf total RNA by RT-PCR using the AccessQuick™ RT-PCR System, then sequenced. (3768)

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Forensic Sci. Int. 152, 89–94. Y-chromosomal STR haplotypes in a Belgian population sample and identification of a micro-variant with a flanking site mutation at DYS19. 2007

De Maesschalck, K,. Vanhoutte, E., Knaepen, K., Vanderheyden, N., Cassiman, J.J., and Decorte, R.

Notes: The authors collected DNA samples from 113 unrelated Belgian males. The PowerPlex® Y System and a GeneAmp® 9700 PCR system were used to amplify 12 Y-chromosome STR loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439). Amplification products were detected using an ABI PRISM® 3100 genetic analyzer and POP-6™ polymer. Allele and haplotype frequencies and haplotype diversity were calculated. A total of 99 different haplotypes were observed. (3655)

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Forensic Sci. Int. 170, 68–72. Allele frequency distribution for 21 autosomal STR loci in Bhutan. 2007

Kraaijenbrink, T., van Driem, G.L., Tshering of Gaselô, K. and de Knijff, P.

Notes: The authors determined the allele frequencies for 21 autosomal STR loci in 936 individuals from the Royal Kingdom of Bhutan using the AmpFlSTR® Identifiler® kit, PowerPlex® 16 System and the F13A01, FESFPS, F13B, LPL (FFFL) Multiplex. DNA was extracted from whole blood samples using the Autopure LS® kit and amplified as directed by the manufacturers. Amplified products were detected using an ABI PRISM® 3100 Genetic Analyzer. (3822)

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