dna-based-fish-species-identification-protocol

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

Isolierung und Expression von rekombinanten Antik&#xF6;rperfragmente an der biologischen Kriegsf&#xFC;hrung Pathogen Brucella melitensis.

To exploit fully the potential of current sequencing technologies for population-based studies, one must enrich for loci from the human genome. Here we evaluate the hybridization-based approach by using oligonucleotide capture probes in solution to enrich for approximately 3.9 Mb of sequence target. We demonstrate that the tiling probe frequency is important for generating sequence data with high uniform coverage of targets. We obtained 93% sensitivity to detect SNPs, with a calling accuracy greater than 99%.

Enrichment of sequencing targets from the human genome by solution hybridization.

Um das Potenzial der aktuellen Technologien f&uuml;r bev&ouml;lkerungsbezogenen Studien vollst&auml;ndig zu nutzen, muss man f&uuml;r Loci von menschlichen Genoms anzureichern. Hier bewerten wir den Hybridisierung-Ansatz mit Oligonucleotide Erfassung Sonden in L&ouml;sung f&uuml;r ca. 3,9 Mb Sequenz Target zu bereichern. Wir zeigen, dass die Fliesen Sonde Frequenz wichtig zur Erzeugung von Sequenzdaten mit hohen gleichm&auml;&szlig;ige Abdeckung der Ziele ist. Wir erhalten 93 % Empfindlichkeit um SNPs, mit einer aufrufenden gr&ouml;&szlig;er als 99 % Genauigkeit zu erkennen.

Bereicherung der Sequenzierung Ziele von menschlichen Genoms von L&ouml;sung Hybridisierung.

The next-generation DNA sequencing workflows require an accurate quantification of the DNA molecules to be sequenced which assures optimal performance of the instrument. Here, we demonstrate the use of qPCR for quantification of DNA libraries used in next-generation sequencing. In addition, we find that qPCR quantification may allow improvements to current NGS workflows, including reducing the amount of library DNA required, increasing the accuracy in quantifying amplifiable DNA, and avoiding amplification bias by reducing or eliminating the need to amplify DNA before sequencing.

Rapid quantification of DNA libraries for next-generation sequencing.

Die n&auml;chste Generation DNA Sequenzierung Workflows erfordern eine genaue Quantifizierung der DNA-Molek&uuml;le zu sequenziert werden die sorgt f&uuml;r eine optimale Leistung des Ger&auml;tes. Hier zeigen wir die Verwendung von qPCR zur Quantifizierung von DNA-Bibliotheken, die in der Sequenzierung der n&auml;chsten Generation verwendet. Au&szlig;erdem finden wir, dass qPCR Quantifizierung Verbesserungen an aktuellen NGS-Workflows, einschlie&szlig;lich der Reduzierung der Bibliothek DNA erforderlich, erh&ouml;hen die Genauigkeit in der amplifiable DNA quantifizieren und Vermeidung von Verst&auml;rkung Voreingenommenheit durch Reduzierung oder Beseitigung der Notwendigkeit zur Verst&auml;rkung von DNA vor der Sequenzierung zulassen kann.

Article	Year
Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis. Journal of immunological methods\| PubMed ID: 12738372	2003
Enrichment of sequencing targets from the human genome by solution hybridization. Genome biology\| PubMed ID: 19835619	2009
Rapid quantification of DNA libraries for next-generation sequencing. Methods (San Diego, Calif.)\| PubMed ID: 20215015	2010

Scott Happe

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