Journal of biomolecular techniques : JBT最新文献

{"title":"PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis.","authors":"Nagesh Kishan Panchal, A. Bhale, Radhika Chowdary, V. Verma, S. S. Beevi","doi":"10.7171/jbt.20-3101-001","DOIUrl":"https://doi.org/10.7171/jbt.20-3101-001","url":null,"abstract":"Formalin-fixed paraffin-embedded (FFPE) tissue specimens have been a staple of research, providing precious resources for molecular and genomic studies. However, the biggest challenge is the extraction of high-quality DNA from FFPE tissues, given that the integrity of DNA is critically affected by formalin fixation. Formaldehyde induces crosslinks in DNA that renders single or double-stranded DNA breaks. Such breaks cause extensive fragmentation that directly influences the quality of DNA purified and the number of templates available for PCR amplification. Thus, protocol for DNA purification from FFPE tissues must effectively extract highly fragmented DNA and reverse cross-linking caused by formalin fixation. DNA extraction methods available in the literature were selected and modified at different stages to optimize a protocol that extracts DNA of sufficient quality and fragment size to be detectable by PCR. Archived FFPE tissues belonged to patients with triple negative breast cancer (TNBC) and benign breast disease were used for the protocol optimization. The best optimized protocol was then used to amplify Exon 4 region of Proviral integration site for Moloney murine leukemia virus1 (Pim1) kinase gene to analyze any probable somatic mutations both in TNBCs and benign breast diseases. Of the 12 different protocols developed, best quality DNA in terms of fragment size and purity was obtained when Tween20 lysis buffer was used for both deparaffinization and overnight digestion along with high salt precipitation. Optimized protocol was then validated by extracting DNAs from 10 TNBCs and 5 benign breast disease specimens with consistent purity and fragment size. PCR amplification and subsequent Sanger's sequencing revealed the presence of mutations in the Exon 4 region of Pim1 kinase. Deparaffinization and overnight digestion in Tween20 lysis buffer along with high salt precipitation yielded the best quality PCR amplifiable DNA for mutational analysis.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87326630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Top-Down ETD-MS Provides Unreliable Quantitation of Methionine Oxidation.","authors":"S. Tadi, J. Sharp","doi":"10.7171/jbt.19-3004-002","DOIUrl":"https://doi.org/10.7171/jbt.19-3004-002","url":null,"abstract":"Methionine oxidation plays a critical role in many processes of biologic and biomedical importance, including cellular redox responses and stability of protein pharmaceuticals. Bottom-up methods for analysis of methionine oxidation can suffer from incomplete sequence coverage, as well as an inability to readily detect correlated oxidation between 2 or more methionines. However, the methodology for quantifying protein oxidation in top-down analyses is lacking. Previous work has shown that electron transfer dissociation (ETD)-based tandem mass spectrometry (MS/MS) fragmentation offers accurate and precise quantification of amino acid oxidation in peptides, even in complex samples. However, the ability of ETD-based MS/MS fragmentation to accurately quantify amino acid oxidation of proteins in a top-down manner has not been reported. Using apomyoglobin and calmodulin as model proteins, we partially converted methionines into methionine sulfoxide by incubation in H2O2. Using top-down ETD-based fragmentation, we quantified the amount of oxidation of various ETD product ions and compared the quantified values with those from traditional bottom-up analysis. We find that overall quantification of methionine oxidation by top-down MS/MS ranges from good agreement with traditional bottom-up methods to vast differences between the 2 techniques, including missing oxidized product ions and large differences in measured oxidation quantities. Care must be taken in transitioning ETD-based quantitation of oxidation from the peptide level to the intact protein level.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78375100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Article Watch: September 2019.","authors":"C. Slaughter","doi":"10.7171/jbt.19-3003-003","DOIUrl":"https://doi.org/10.7171/jbt.19-3003-003","url":null,"abstract":"This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Avenue, Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84384859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Survey on Scientific Shared Resource Rigor and Reproducibility.","authors":"Kevin L Knudtson, Robert H Carnahan, Rebecca L Hegstad-Davies, Nancy C Fisher, Belynda Hicks, Peter A Lopez, Susan M Meyn, Sheenah M Mische, Frances Weis-Garcia, Lisa D White, Katia Sol-Church","doi":"10.7171/jbt.19-3003-001","DOIUrl":"10.7171/jbt.19-3003-001","url":null,"abstract":"<p><p>Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members <i>via</i> a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (<i>i.e.</i>, those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.</p>","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"30 3","pages":"36-44"},"PeriodicalIF":0.0,"publicationDate":"2019-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657953/pdf/jbt.30-36.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41224806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive Detection of Germline and Somatic Structural Mutation in Cancer Genomes by Bionano Genomics Optical Mapping.","authors":"A. Pang, J. Lee, T. Anantharaman, E. Lam, A. Hastie, M. Borodkin","doi":"10.1158/1538-7445.SABCS18-5122","DOIUrl":"https://doi.org/10.1158/1538-7445.SABCS18-5122","url":null,"abstract":"In cancer genetics, the ability to identify constitutive and low-allelic fraction structural variants (SVs) is crucial. Conventional karyotype and cytogenetics approaches are manually intensive. Microarrays and short-read sequencing cannot detect calls in segmental duplications and repeats, often miss balanced variants, and have trouble finding low-frequency mutations. We describe the use of Bionano Genomics Saphyr platform to comprehensively identify SVs for studying cancer genomes. DNA >100 kbp is extracted, labelled at specific motifs, and linearized through NanoChannel arrays for visualization. Molecule images are digitized and de novo assembled, creating chromosomal arm scale genome maps. Somatic mutations can be identified by running the variant annotation pipeline that compares the cancer sample assembly SVs against >600,000 SVs in Bionano control sample SV database, and against a matched control sample SVs, if avaliable. Also, two new Bionano pipelines leverage these long molecules to identify additional somatic SVs: the copy number variation (CNV) and the molecule mapping pipelines. By examining the coverage-depth of molecules alignment to the public reference, the pipeline can identify megabases long CNVs. Similarly, clusters of split-molecule alignments can reliably find translocations and other rearrangements. We applied this suite of discovery tools to identify SVs in a well-studied melanoma cell line COLO829. We collected data from the tumor and the matched blood cell line, constructed contiguous assemblies (N50 >50 Mbp), and called >6,000 SVs in each genome. Then, we classified 51 as somatic by comparing the tumor and the blood control. The two new pipelines further increased sensitivity to rearrangements, for example they captured a BRAF duplication, and other chromosome-arm CNVs. We apply these thorough approaches to multiple well-studied cancer lines to identify novel SVs missed by previous studies. In conclusion, with one comprehensive platform, Saphyr can discover a broad range of traditionally refractory but relevant SVs, and further improves our understanding of cancer.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"48 1","pages":"S9"},"PeriodicalIF":0.0,"publicationDate":"2019-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88496108","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Article Watch: July 2019.","authors":"C. Slaughter","doi":"10.7171/jbt.19-3002-002","DOIUrl":"https://doi.org/10.7171/jbt.19-3002-002","url":null,"abstract":"This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Avenue, Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"97 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80521031","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Article Watch: April 2019.","authors":"C. Slaughter","doi":"10.7171/jbt.19-3001-003","DOIUrl":"https://doi.org/10.7171/jbt.19-3001-003","url":null,"abstract":"This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"4 1","pages":"12-18"},"PeriodicalIF":0.0,"publicationDate":"2019-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84792322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signal Analysis of Nanopore RNA Sequencing to Interrogate Poly(A) Tails and Post-Transcriptional Modifications.","authors":"Roham Razaghi","doi":"10.1016/J.BPJ.2018.11.1935","DOIUrl":"https://doi.org/10.1016/J.BPJ.2018.11.1935","url":null,"abstract":"","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"30 1","pages":"S60"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90062117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Measuring the Stoichiometry of Antimicrobial Peptides in Nanodiscs with Native Mass Spectrometry.","authors":"M. Marty, L. Walker, E. Marzluff","doi":"10.1016/J.BPJ.2018.11.504","DOIUrl":"https://doi.org/10.1016/J.BPJ.2018.11.504","url":null,"abstract":"","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"15 1","pages":"S55"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84378093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microbes in Space.","authors":"K. Venkateswaran","doi":"10.1201/B22230-43","DOIUrl":"https://doi.org/10.1201/B22230-43","url":null,"abstract":"In light of an upcoming new era of human expansion in the universe, such as future space travel to Mars, the microbiome of the closed space environment needs to be examined thoroughly to identify the types of microorganisms that can accumulate in this unique environment, how long they persist and survive and their impact on human health and spacecraft infrastructure. As part of this NASA initiative, the viable microbial communities on ISS surfaces from eight different locations over three flight missions, spanning 14 months, were characterized using culture-based techniques, qPCR and amplicon sequencing of the 16S rRNA gene and internal transcribed spacer region using the Illumina platform. Across three flight samplings, K. pneumoniaereads, an opportunistic BSL-2 pathogen, were retrieved during Flight 1 and successively its reads persisted in Flight 2. Subsequently, in Flight 3, most of the locations were inflicted with the presence of this opportunistic pathogen. Other noticeable opportunistic pathogens of all flights were A. baumannii, E. cloacae, S. enterica,and S. sonneias well as some fungi. None of the pathogenic fungi were persistent in any of the locations sampled.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"69 1","pages":"S56"},"PeriodicalIF":0.0,"publicationDate":"2018-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78815720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}