Method For Sequence Determination Research Articles

DuBA.flow─A Low-Cost, Long-Read Amplicon Sequencing Workflow for the Validation of Synthetic DNA Constructs.

Modern biological science, especially synthetic biology, relies heavily on the construction of DNA elements, often in the form of plasmids. Plasmids are used for a variety of applications, including the expression of proteins for subsequent purification, the expression of heterologous pathways for the production of valuable compounds, and the study of biological functions and mechanisms. For all applications, a critical step after the construction of a plasmid is its sequence validation. The traditional method for sequence determination is Sanger sequencing, which is limited to approximately 1000 bp per reaction. Here, we present a highly scalable in-house method for rapid validation of amplified DNA sequences using long-read Nanopore sequencing. We developed two-step amplicon and transposase strategies to provide maximum flexibility for dual barcode sequencing. We also provide an automated analysis pipeline to quickly and reliably analyze sequencing results and provide easy-to-interpret results for each sample. The user-friendly DuBA.flow start-to-finish pipeline is widely applicable. Furthermore, we show that construct validation using DuBA.flow can be performed by barcoded colony PCR amplicon sequencing, thus accelerating research.

Bacterial community dynamics during different stages of processing of smoked bacon using the 16S rRNA gene amplicon analysis

To identify the microbial community and origin of the spoilage flora of bacon, the changes in microbial population numbers and community structure were followed along the processing line, using culture-independent and culture-dependent methods. 16S rRNA gene amplicon sequencing (16S-seq) analysis showed that community complexity and structure significantly differed at different processing stages. Some 428 bacterial groups were ascertained at genus level, and Acinetobacter, Pseudomonas, Psychrobacter, and Brochothrix were the predominant bacteria on raw meats. After curing specimens dominated by Psychrobacter, Weissella, Vibrio, Leuconostoc, Myroides, Acinetobacter, and Lactobacillus, a total of 33 species were identified by traditional microbiological analyses and direct sequence determination methods. Our results indicated that curing should be considered one of the primary factors during various processing steps, presumably contaminating the products directly or indirectly.

Expansion of a highly sensitive and specific ELISA test for diagnosis of hydatidosis using recombinant EgB8/2 protein.

Objective(s):Hydatidosis is a zoonotic infection and endemic in Iran. Due to the serological cross-reactivity (of sera) with other parasitic infection, diagnosis of hydatid cyst is considered to be problematic. In this regard, application of recombinant antigens improves serological diagnosis for human hydatidosis. Here, we present an ELISA test based on B8/2 recombinant antigen of Echinococcus granulosus with particular regard to its capability to diagnose human hydatidosis.Materials and Methods:The synthesized E. granulosus B8/2 (EgB8/2) gene was sub-cloned into pET28b (+) plasmid. Nde1 and Hind3 restriction enzymes were used to confirm the recombinant plasmid extraction. Cloning was verified by colony PCR, digestion enzymes, and sequence determination methods. To express rtEgB8/2, strains of Escherichia coli BL21 (DE3) pLysS and Rosetta (DE3) were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). A Ni-NTA column was used for purification, and the expressed protein was analyzed by SDS-PAGE as well as western blotting. ELISA test was used to identify the antigenicity of produced protein.Results:The presence of EgB8/2 gene fragment in the recombinant plasmid was confirmed. SDS-PAGE showed that the BL21 (DE3) pLysS strain had the highest level of expression and a protein band of 11 kDa was observed in induced bacteria. Western blotting approved the purity of rtEgB8/2 protein, and ELISA test measured sensitivity and specificity as 95% and 97.5%, respectively.Conclusion: E. granulosus metacestode contains a high amount of antigen B protein. These results confirm the reproducibility of high-quality rtEgB8/2 recombinant antigen as a reliable candidate in serological test.

Facile method to sequence cyclic peptides/peptoids via one-pot ring-opening/cleavage reaction.

A facile method for sequence determination of cyclic peptides/peptoids is described. Macrocyclic peptides/peptoids of 3-10 residues were efficiently synthesized through thioether formation. One-pot reaction of thioether-embedded cyclic peptides/peptoids involving cyanogen bromide-mediated ring-opening and cleavage provides linearized molecules, which can be efficiently sequenced by tandem mass spectrometry.

High-Throughput Sequence Determination of Cyclic Peptide Library Members by Partial Edman Degradation/Mass Spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.

5470707 Hydrogen bond labeling and base sequence determination methods for DNA or RNA : Sasaki Yuj; Harada Yoshinor Kawagoe, Japan assigned to Hitachi Ltd

5470707 Hydrogen bond labeling and base sequence determination methods for DNA or RNA : Sasaki Yuj; Harada Yoshinor Kawagoe, Japan assigned to Hitachi Ltd

5470707 Hydrogen bond labeling and base sequence determination methods for DNA or RNA : Sasaki Yuj; Harada Yoshinor Kawagoe, Japan assigned to Hitachi Ltd

5470707 Hydrogen bond labeling and base sequence determination methods for DNA or RNA : Sasaki Yuj; Harada Yoshinor Kawagoe, Japan assigned to Hitachi Ltd

Enzymatic digestion on the sample foil as a method for sequence determination by plasma desorption mass spectrometry: the primary structure of porpoise relaxin

Plasma desorption mass spectrometry (PDMS) was used to determine the C-terminus of the B-chain porpoise relaxin, a polypeptide hormone having three dimensional and disulfide homology with insulin. Trypsin was used to generate a series of peptide fragments for mapping by PDMS. The C-terminal fragments were purified and their sequences determined by on-foil digestion with carboxypeptidase Y.

11] 16 S ribosomal RNA cataloging

Publisher Summary This chapter discusses 16 S ribosomal RNA cataloging. Phylogenetic studies on cyanobacteria and chloroplasts using the 16 S ribosomal RNA (rRNA) cataloging approach as a tool have been done by two different methods: the traditional one which is generally not used anymore and the advanced technique which is presented in detail in chapter. rRNA can be isolated either from ribosomal subunits or from crude extracts. Experience with more than 450 prokaryotic species has shown that the following steps will lead in most cases to satisfactory amounts and purity of 16 S rRNA. The original methods of sequence determination have been improved to facilitate sequencing of the more than 70 oligonucleotides, varying in length from 6 to 20 nucleotides, making up a 16 S rRNA catalog. These changes include the replacement of the “Homomixes” by ammonium formate gradient chromatography, the use of alkaline hydrolysis rather than enzymatic cleavage for the generation of subfragments, and the introduction of additional steps to improve analysis of those oligonucleotides whose sequence could not be determined unambiguously after the secondary analysis.

13] Inositol-containing sphingolipids

Publisher Summary Glycophosphosphingolipids (GPSs) are membrane glycolipids containing a phosphodiester linkage between inositol and ceramide. The structure, “inositol (1-O)-phosphoryl-(O-1)ceramide” constitutes a common core. This chapter discusses some known glycophosphophingolipids. There is also a discussion of the procedure of isolation of a glycophosphosphingolipid fraction from plants. There is a description of the production of a carboxyl-reduced oligosaccharide mixture from the glycophosphoceramide concentrate. To isolate major fractions of the oligosaccharides, gel permeation chromatography on Bio-Rad P gels is used, effectively separating oligo saccharide fractions up to 10 sugars in length. Several methods in high-performance liquid chromatography (HPLC) can be utilized for further purification of the oligosaccharides, including reversed-phase separation of the acetylated derivatives and amino-derivatized normal-phase chromatography on silica gel. Detection methods include (1) benzoylation of the amino group on the glucosamine, (2) use of fluorescamine or o-phthaldehyde to synthesize a fluorophore on the glucosamine in glucosamine-containing GPSs for fluorescence detection of the molecules, and (3) use of tritiated acetic anhydride for radiolabeling of the aminosugar. High-performance thin-layer chromatography can complement these methods. There is also some discussion in the chapter about the new methods of sequence determination using fast atom bombardment mass spectrometry.

Determination of polypeptide amino acid sequences from the carboxyl terminus using angiotensin I converting enzyme.

A method for sequence analysis of polypeptides starting at the carboxyl terminus is described that utilizes degradation of the polypeptide into dipeptides with angiotensin I converting enzyme. Dipeptides were identified by gas chromatography-mass spectroscopy. Dipeptide alignment was achieved by replicate digestion of the polypeptide after modification at the carboxyl terminus either by chemical or enzymatic removal of one residue or by addition of a single residue. The addition reaction involved coupling of L-alpha-aminobutyric acid under conditions described herein which yielded essentially complete conversions. Unlike sequence determination methods that commence from the polypeptide amino terminus, this procedure does not require that a polypeptide have a free amino terminus for successful application. A number of polypeptides with varying chain lengths (up to 49 residues), containing among them most of the common amino acids, have been successfully analyzed in amounts as low as 5 nmol.

A new approach for the solid phase sequence determination of proteins

The use of the modified Edman reagent, 4-N,Ndimethylaminoazobenzene 4’-isothiocyanate, in the stepwise degradation of peptides and proteins has provided a useful method for sequence determination [l-3] . Released N-terminal amino acids are identified as 4-N,N-dimethylaminoazobenzene 4’-thiohydantoins directly by thin-layer chromatography (t.1.c.) on polyamide sheets, the intense colour of the amino acid derivatives allowing detection in subnanomol quantities. Low repetitive yields due to extraction losses and time-consuming operations at each drying step have reduced both sensitivity and efficiency of the manual sequencing work. However, the solid phase degradation of Laursen [4,5] whereby peptides are attached covalently to an insoluble support, eliminates extraction losses and shortens greatly the time needed for the drying of residues after the extractions. A solid phase degradation procedure using 4-N,Ndimethylaminoazobenzene 4’-isothiocyanate has been developed. The N-terminal sequence of native lysozyme (first 19 residues) and glucagon (first 10 residues) attached to controlled porosity glass beads activated by p-phenylene diisothiocyanate have been obtained. Reduced Histidinol dehydrogenase (mol. wt 40 000) from Salmonella typhimwium, of hitherto unknown sequence has been attached via cysteine residues to an iodoacetamide derivative of controlled porosity glass beads and the first eight amino acid residues determined. This new solid phase sequenching method is sensitive and efficient, requiring practically, 210 nmol protein as the starting material. Each cycle of

Determination de la structure primaire de la proteine du virus de la mosaïque de la luzerne (souche s) II. Sequence complete de la proteine

Determination of the primary structure of alfalfa mosaic virus (strain S) coat protein. II. Complete sequence of the protein The alfalfa mosaic virus protein was submitted to the action of cyanogen bromide. Four peptides were isolated. Study of these peptides allowed us to determine the order. Then protein was submitted, after S-carboxymethylation or S-aminoethylation, to the action of different proteolytic enzymes: trypsin, chymotrypsin, thermolysin and papain. The peptides issued from these different hydrolysis were separated on Dowex 50 X4 and Dowex 1 X 2, and their amino acid composition was determined. The use of classical methods of sequence determination, of mass spectrometry and for one case the use of a sequencer, lead to the obtention of the primary structure of all the tryptic peptides. The studies of chymotryptic, thermolytic and papainic hydrolysates, and of cyanogen bromide rupture, allowed us to isolate the overlapping peptides which were necessary for the reconstitution of the complete proteic chain.

A rellable mapping method for sequence determination of oligodeoxyribonucleotides by mobility shift analysis

The method for sequence analysis of large oligodeoxyribonucleotides based on the characteristic mobility shifts of their sequential partial degradation products on two-dimensional homochromatography has been perfected using a large number of synthetic oligodeoxyribonucleotides of defined sequences as standards. Flat bed electrophoresis with careful temperature control gave entirely reproducible mobilities in the first dimension. Using this information, an accurate formula has been derived for calculating the relative electrophoretic mobilities of oligodeoxyribonucleotides of any composition. This formula is used to calculate the mobility shifts between two consecutive oligodeoxyribonucleotides in a series of partial products of an unknown oligomer distributed in the two-dimensional homochromatogram which differ by one nucleotide in length. This is compared with the observed mobility shift value to identify the added nucleotide. This provides a direct and rapid method for obtaining the unambiguous sequence of an entire oligodeoxyribonucleotide up to 15 nucleotides in length.

Solid Phase Peptide Synthesis

Abstract The last two decades have been an era of rapid progress in peptide research. This era was begun by the work of Sanger on the amino acid sequence determination of insulin and by du Vigneaud on the structure determination and synthesis of oxytocin. This period has seen impressive progress in the structure elucidation and synthesis of many peptides of natural origin and of great biological significance, as well as in methods for sequence determination and chemical synthesis of peptides [1–4]. Perfection of techniques and instruments for automatic determination of the amino acid sequence of peptides and proteins has made possible a greatly broadened understanding of genetics and evolution as well as the more chemical areas of mechanism of action of enzymes and hormones, and physical chemistry of peptides and proteins. Effective methods of peptide synthesis are crucial to progress in this area, because only by synthesis can adequate amounts of important peptides be made available for chemical, biological, and physical studies, as well as for exploration of the structure-function aspects of biological molecules. In general, progress in peptide synthesis has lagged far behind that in amino acid sequence determination. This is not surprising since effective peptide synthesis requires a very sophisticated system of selectively removable protecting groups for functions of the amino acids involved, and the synthesis of a large heteropolytner of defined sequence requires near perfection of each one of the many steps of the assembly. The classical approach to peptide synthesis, using standard organic chemical methods of synthesis and purification of intermediates, has yielded impressive results during these two decades. However, the special problems associated with the assembly of large molecules make staggering investments in time and materials necessary for the synthesis of large peptides or proteins by classical methods.

A simple and highly sensitive method for sequence determination of 32P-labeled oligonucleotides

A new procedure is described for the sequence determination of oligonucleotides produced by digestion of RNA with pancreatic RNase A. The oligonucleotide is treated with spleen exonuclease and all intermediates are resolved by thin-layer chromatography on polyethyleneimine plates. On the basis of the increase in mobility it can be decided for each successive step whether a Gp- or an Ap-residue has been removed by reference to a calibration grid. The method is very simple and can easily be applied to a large number of samples. An amount of 32P-radioactivity corresponding to 40 dpm/nucleotide is sufficient for analysis.

1H NMR peptide sequential analysis using superconducting magnets

The principle, scope, and limitations of methods for sequence determination of oligopeptides by variation of pH with and without addition of lanthanide salt shift reagents (L sR) are discussed. Although the latter method seems very promising for aqueous solutions in general, extensive line broadening in alkaline medium poses serious limitation to its applicability. The use of macrocyclic reagents to induce shifts (MIS) is proposed as a further promising technique.

The Primary Structure of the Porcine Pancreatic Secretory Trypsin Inhibitor I: AMINO ACID SEQUENCE OF THE REDUCED S-AMINOETHYLATED PROTEIN

Abstract The amino acid sequence of porcine pancreatic secretory trypsin inhibitor I (Greene, L. J., Dicarlo, J. J., Sussman, A. J., Bartelt, D. C., and Roark, D. E., J. Biol. Chem., 243, 1804 (1968)) was determined by a combination of selective trypsin and chymotrypsin hydrolysis reactions and conventional methods for sequence determination of small peptides. Arginine-directed cleavage was achieved by blocking lysyl and S-2-aminoethylcysteinyl residues with maleic anhydride. Hydrolysis of lysyl bonds in peptides containing S-2-aminoethylcysteine was accomplished by trypsin at pH 11.0. All of the overlap information required to order the small peptides was obtained by the identification of the amino- and carboxy-terminal residues of the products at each stage of the degradative procedure. The yields of the peptides, 38 to 93%, compared favorably with those obtained by the conventional complete trypsin hydrolysis of the bovine pancreatic secretory trypsin inhibitor, a molecule of the same size and similar structure. The amino acid sequence of the porcine inhibitor has been shown to be [see PDF for sequence]. This sequence exhibits 12 differences in 56 amino acid residues per molecule from that of the bovine pancreatic secretory trypsin inhibitor (Greene, L. J., and Bartelt, D. C., J. Biol. Chem., 244, 2646 (1969)).

The amino acid sequence of Clostridium butyricum ferredoxin

The amino acid sequence of Clostridium butyricum ferredoxin, determined by the application of conventional methods of sequence determination primarily to the S-β-aminoethyl protein, was found to be: Ala-Phe-Val-Ile-Asn-Asp-Ser-Cys-Val-Ser- Cys-Gly-Ala-Cys-Ala-Gly-Glu-Cys-Pro-Val-Ser-Ala-Ile-Thr-Gln-Gly-Asp-Thr-Gln- Phe-Val-Ile-Asp-Ala-Asp-Thr-Cys-Ile-Asp-Cys-Gly-Asn-Cys-Ala-Asn-Val-Cys-Pro- Val-Gly-Ala-Pro-Asn-Gln-Glu. This sequence differs in nine positions from that of Clostridium pasteurianum ferredoxin. The positions of the cysteine and proline residues are invariant in these two species, as is the sequence including residues 30–52.

The amino acid sequence of Clostridium butyricum ferredoxin.

Abstract The amino acid sequence of Clostridium butyricum ferredoxin, determined by the application of conventional methods of sequence determination primarily to the S-β-aminoethyl protein, was found to be: Ala-Phe-Val-Ile-Asn-Asp-Ser-Cys-Val-Ser- Cys-Gly-Ala-Cys-Ala-Gly-Glu-Cys-Pro-Val-Ser-Ala-Ile-Thr-Gln-Gly-Asp-Thr-Gln- Phe-Val-Ile-Asp-Ala-Asp-Thr-Cys-Ile-Asp-Cys-Gly-Asn-Cys-Ala-Asn-Val-Cys-Pro- Val-Gly-Ala-Pro-Asn-Gln-Glu. This sequence differs in nine positions from that of Clostridium pasteurianum ferredoxin. The positions of the cysteine and proline residues are invariant in these two species, as is the sequence including residues 30–52.