Applied Biosystems

About: Applied Biosystems is a based out in . It is known for research contribution in the topics: Mass spectrometry & Capillary electrophoresis. The organization has 1521 authors who have published 1579 publications receiving 285423 citations.

Sequencing Multimegabase-Template DNA with BigDye Terminator Chemistry

Abstract: In microbial genome or large-insert clone sequencing projects that use the predominant random subclone sequencing strategy, progress tends to decrease dramatically at late stages as one confronts gaps. At these points, DNA is under-represented or unstable in subclones (E.Y. Chen et al. 1996; Chissoe et al. 1997). Further sequencing with additional random subclones is then inefficient at best, and one must frequently employ alternative cloning systems or additional methods like long-range PCR to recover missing DNA (C.N. Chen et al. 1996). The variability of performance of these methods and the necessity for custom-tailored work tend to hamper the late stages of sequencing efforts. In contrast, if one can sequence directly from genomic DNA (or large-insert clones such as BACs or PACs) with walking primers, cumbersome work to fill gaps could be completed in a much shorter time. As an example, in a recent project to sequence the 750-kb genome of Ureaplasma urealyticum (J. Glass, in prep.) assemblage of ∼13,000 sequence reads and combinatorial PCR reactions to join contigs left two gaps. No λ pUC, or M13 subclones were recovered that spanned the gaps, nor were PCR products derived with any of several sets of flanking primers. The difficulty of cloning these segments is probably attributable to repeated sequences in and near the two gaps, but the high sensitivity of the recently introduced BigDye terminator (Rosenblum et al. 1997) permitted direct sequencing of the gap regions on genomic U. urealyticum DNA templates. Using the conditions described in this report, two gaps of 259 and 121 bp were sequenced from both strands with walking primers to complete the project of 751,723 bp. Direct sequencing was further tested for larger templates, and good results were reproducibly obtained with 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli genomic DNA (see example in Fig. ​Fig.1).1). In addition, several difficult gaps in sequencing projects with BAC clones, ranging in size from 140 to 250 kb, have also been filled in this manner. Essentially the method is applicable whenever 2–3 μg of high-quality large-template DNA is available. Figure 1 Sequencing of E. coli K12 strain genomic DNA with BigDye terminators. Approximately 3 μg of E. coli DNA was sequenced with an apaG gene primer (5′-GTTCCCACACTCATTCATTA) using the conditions described in the text.

PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5' nuclease (TaqMan) assay

Abstract: Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E coli (EHEC) eaeA gene In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E coli O157:H7 DNA The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E coli O157:H7 when >/=10(3) CFU/ml was present in modified tryptic soy broth (mTSB) or modified E coli broth and when >/=10(4) CFU/ml was present in ground beef-mTSB mixtures Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >/=10(2) CFU/ml Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >/=10(4) CFU/ml was present in the sample Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E coli O157:H7 in ground beef and potentially in other food and environmental samples

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (bn-page)

Abstract: Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b6f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.