Pub Date : 2021-01-03DOI: 10.1101/2020.12.26.20248878
T. Brown, Katherine S. Schaefer, A. Tsang, H. Yi, J. Grimm, Andrew L. Lemire, Fadi M. Jradi, Charles Kim, Kevin McGowan, K. Ritola, D. Armstrong, H. Mostafa, Wyatt L. Korff, Ronald D. Vale, L. Lavis
The worldwide COVID-19 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce two pH sensitive ''LAMPshade'' dyes as novel readouts in an isothermal RT-LAMP amplification assay for SARS-CoV-2 RNA. The resulting JaneliaLAMP (jLAMP) assay is robust, simple, inexpensive, has low technical requirements and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.
{"title":"Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes","authors":"T. Brown, Katherine S. Schaefer, A. Tsang, H. Yi, J. Grimm, Andrew L. Lemire, Fadi M. Jradi, Charles Kim, Kevin McGowan, K. Ritola, D. Armstrong, H. Mostafa, Wyatt L. Korff, Ronald D. Vale, L. Lavis","doi":"10.1101/2020.12.26.20248878","DOIUrl":"https://doi.org/10.1101/2020.12.26.20248878","url":null,"abstract":"The worldwide COVID-19 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce two pH sensitive ''LAMPshade'' dyes as novel readouts in an isothermal RT-LAMP amplification assay for SARS-CoV-2 RNA. The resulting JaneliaLAMP (jLAMP) assay is robust, simple, inexpensive, has low technical requirements and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"35 1","pages":"121-133"},"PeriodicalIF":0.0,"publicationDate":"2021-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91151414","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}
Pub Date : 2021-01-02DOI: 10.1101/2020.12.26.20248880
Albert D. Yu, Kristina Galatsis, Jian Zheng, Jasmine Quynh Le, Dingbang Ma, Stanley Perlman, M. Rosbash
Conventional reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has struggled to fulfill the unprecedented need for diagnostic testing created by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Complexity and cost hinder access to testing, and long turnaround-time decreases its utility. To ameliorate these issues, we focus on saliva and introduce several advances to colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technology; RT-LAMP offers a minimal equipment alternative to RT-qPCR. First, we validated the use of the novel dye LAMPShade Violet (LSV), which improves the visual clarity and contrast of the colorimetric readout. Second, we compared different inactivation conditions on infectivity and RNA yield from saliva. Third, we developed a ten-minute RNA purification protocol from saliva. We call this magnetic bead protocol SalivaBeads. Finally, we developed a magnetic stick, StickLAMP, which provides reliable bead-based RNA purification as well as simple and low-cost access to scalable testing from saliva.
{"title":"Development of a saliva-optimized RT-LAMP assay for SARS-CoV-2","authors":"Albert D. Yu, Kristina Galatsis, Jian Zheng, Jasmine Quynh Le, Dingbang Ma, Stanley Perlman, M. Rosbash","doi":"10.1101/2020.12.26.20248880","DOIUrl":"https://doi.org/10.1101/2020.12.26.20248880","url":null,"abstract":"Conventional reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has struggled to fulfill the unprecedented need for diagnostic testing created by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Complexity and cost hinder access to testing, and long turnaround-time decreases its utility. To ameliorate these issues, we focus on saliva and introduce several advances to colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technology; RT-LAMP offers a minimal equipment alternative to RT-qPCR. First, we validated the use of the novel dye LAMPShade Violet (LSV), which improves the visual clarity and contrast of the colorimetric readout. Second, we compared different inactivation conditions on infectivity and RNA yield from saliva. Third, we developed a ten-minute RNA purification protocol from saliva. We call this magnetic bead protocol SalivaBeads. Finally, we developed a magnetic stick, StickLAMP, which provides reliable bead-based RNA purification as well as simple and low-cost access to scalable testing from saliva.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"70 1","pages":"102-113"},"PeriodicalIF":0.0,"publicationDate":"2021-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86306023","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}
Pub Date : 2020-10-04DOI: 10.1101/2020.10.01.20205492
J. Ongerth, R. Danielson
The project purpose was to examine ability of a loop-mediated isothermal amplification (LAMP) assay to quantify SARS-CoV-2 in raw sewage, directly, using no preliminary sample processing for virus concentration and RNA extraction. An objective was to take advantage of extensive recently published work to facilitate process development, principally primer selection, and to use readily available off the shelf materials with conventional lab procedure and equipment. Well-developed and referenced primers for ORF1a, E, and N-gene targets were selected and applied, using commercially available synthetic RNA standards, and raw sewage from a local wastewater agency serving 650,000. County health department monitoring provided current COVID-19 data. Testing defined performance characteristics for each primer set, with significant differences between them. Specific amplification of SARS-CoV-2 RNA was observed using each of the primer sets, with E-gene and N-gene primers most effective. Positive analysis results from all raw sewage samples corresponded to calculated concentrations of virus in 5-10 L raw sewage aliquots for 25 L reactions. Results show that even at low reported case rates e.g. 1-10/100,000, SARS-CoV-2 is present in raw sewage at > 1-5/ L, permitting direct LAMP-based detection. Use of RT qLAMP will facilitate wastewater-based epidemiology as an important component for COVID-19 control.
{"title":"RT qLAMP--Direct Detection of SARS-CoV-2 in Raw Sewage","authors":"J. Ongerth, R. Danielson","doi":"10.1101/2020.10.01.20205492","DOIUrl":"https://doi.org/10.1101/2020.10.01.20205492","url":null,"abstract":"The project purpose was to examine ability of a loop-mediated isothermal amplification (LAMP) assay to quantify SARS-CoV-2 in raw sewage, directly, using no preliminary sample processing for virus concentration and RNA extraction. An objective was to take advantage of extensive recently published work to facilitate process development, principally primer selection, and to use readily available off the shelf materials with conventional lab procedure and equipment. Well-developed and referenced primers for ORF1a, E, and N-gene targets were selected and applied, using commercially available synthetic RNA standards, and raw sewage from a local wastewater agency serving 650,000. County health department monitoring provided current COVID-19 data. Testing defined performance characteristics for each primer set, with significant differences between them. Specific amplification of SARS-CoV-2 RNA was observed using each of the primer sets, with E-gene and N-gene primers most effective. Positive analysis results from all raw sewage samples corresponded to calculated concentrations of virus in 5-10 L raw sewage aliquots for 25 L reactions. Results show that even at low reported case rates e.g. 1-10/100,000, SARS-CoV-2 is present in raw sewage at > 1-5/ L, permitting direct LAMP-based detection. Use of RT qLAMP will facilitate wastewater-based epidemiology as an important component for COVID-19 control.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"719 1","pages":"206-213"},"PeriodicalIF":0.0,"publicationDate":"2020-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74765924","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}
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.
本专栏重点介绍本出版物的读者感兴趣的最近发表的文章。我们鼓励ABRF成员将他们认为重要和有用的文章信息转发给Clive Slaughter, MCG-UGA医疗合作伙伴，1425 Prince Ave.， Athens, GA 30606, USA。电话:(706)713-2216;传真:(706)713-2221;电子邮件;cslaught@uga.edu，或任何编委会成员。文章摘要反映的是审稿人的意见，而不一定是协会的意见。
{"title":"Article Watch: October, 2020.","authors":"","doi":"10.7171/jbt.20-3103-006","DOIUrl":"https://doi.org/10.7171/jbt.20-3103-006","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":"37 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83650235","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}
Pub Date : 2020-08-01DOI: 10.1002/9783527678679.dg09917
P. Jagtap
The Proteomics Research Group initiated a study in 2018 to facilitate the adoption of data-independent acquisition (DIA) by providing participants with generic methods and test samples containing a HeLa digest spiked with different levels of four non-endogenous proteins. This resulted in the generation of 49 participant datasets submitted by laboratories across the world (20 countries and 16 states in the U.S.). The files from two laboratories were selected for further analysis using varied library approaches. The data (which will be made available via MassIVE) can be used by software developers or experts evaluating performance with different acquisition settings in addition to DIA newcomers to help them implement the technique in their laboratory.
{"title":"PRG.","authors":"P. Jagtap","doi":"10.1002/9783527678679.dg09917","DOIUrl":"https://doi.org/10.1002/9783527678679.dg09917","url":null,"abstract":"The Proteomics Research Group initiated a study in 2018 to facilitate the adoption of data-independent acquisition (DIA) by providing participants with generic methods and test samples containing a HeLa digest spiked with different levels of four non-endogenous proteins. This resulted in the generation of 49 participant datasets submitted by laboratories across the world (20 countries and 16 states in the U.S.). The files from two laboratories were selected for further analysis using varied library approaches. The data (which will be made available via MassIVE) can be used by software developers or experts evaluating performance with different acquisition settings in addition to DIA newcomers to help them implement the technique in their laboratory.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"48 1","pages":"S39"},"PeriodicalIF":0.0,"publicationDate":"2020-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77619793","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}
Yamna Khurshid, M. Saeed, Jerika T. Lam, S. Simjee, Z. Haq, Aftab Ahmed, Allis Chien, Roy Martin
Medicinal plants are rich source of pharmaceutically active peptides and proteins. Momordica charantia, a traditional medicinal plant commonly known as bitter melon, is a member of a family Cucurbitaceae and has been explored for various human diseases such as diabetes, peptic ulcer, malaria, infectious diseases, and cancer. Yet, to date only a handful of studies are available related to the therapeutic potential of Momordica charantia seed proteins. In present study, a novel trypsin inhibitor McTI was purified by 2D-LC technique from ammonium sulphate precipitated crude seed proteins of Momordica charantia. The crude seed proteins and gel filtration fractions were explored for their cytotoxic response against MDA-MB-231 cells. Later, for deeper understanding, complete amino acid sequence of McTI was established by Edman protein sequencing and 3D structure was predicted by comparative homology modeling using template trypsin inhibitor 3 from spiny bitter cucumber. In silico molecular docking and dynamic simulation experiments were also performed to study the interaction of McTI with bovine trypsin. The results revealed that McTI is a 30 amino acids peptide having a molecular mass of 3388.4 Da and showed sequence similarity with previously reported cystine knot trypsin inhibitors from plant. McTI is a disulfide rich peptide having 6 cysteine residues that can form 3 disulfide bonds (Cys3-Cys20, Cys10-Cys22 and Cys16-Cys28). Six hydrogen bond interactions of McTI with bovine trypsin were observed in molecular docking whereas, additional hydrogen bond interactions were noticed in molecular dynamic simulation studies. In cytotoxicity analysis against MDA-MB-231 cells, crude seed proteins and F12 exhibited a significant dose dependent response with an IC 50 values of 82.10 ± 6.46 µg/ml and 81.13 ± 4.26, respectively. These findings suggest Momordica charantia proteins as a possible anticancer agent. Furthermore, McTI is a valuable addition in the squash inhibitor family.
{"title":"Molecular Characterization and Cytotoxic Activity of McTI, a Novel Cystine-Knot Inhibitor from Momordica charantia.","authors":"Yamna Khurshid, M. Saeed, Jerika T. Lam, S. Simjee, Z. Haq, Aftab Ahmed, Allis Chien, Roy Martin","doi":"10.1175/JbtAbstract","DOIUrl":"https://doi.org/10.1175/JbtAbstract","url":null,"abstract":"Medicinal plants are rich source of pharmaceutically active peptides and proteins. Momordica charantia, a traditional medicinal plant commonly known as bitter melon, is a member of a family Cucurbitaceae and has been explored for various human diseases such as diabetes, peptic ulcer, malaria, infectious diseases, and cancer. Yet, to date only a handful of studies are available related to the therapeutic potential of Momordica charantia seed proteins. In present study, a novel trypsin inhibitor McTI was purified by 2D-LC technique from ammonium sulphate precipitated crude seed proteins of Momordica charantia. The crude seed proteins and gel filtration fractions were explored for their cytotoxic response against MDA-MB-231 cells. Later, for deeper understanding, complete amino acid sequence of McTI was established by Edman protein sequencing and 3D structure was predicted by comparative homology modeling using template trypsin inhibitor 3 from spiny bitter cucumber. In silico molecular docking and dynamic simulation experiments were also performed to study the interaction of McTI with bovine trypsin. The results revealed that McTI is a 30 amino acids peptide having a molecular mass of 3388.4 Da and showed sequence similarity with previously reported cystine knot trypsin inhibitors from plant. McTI is a disulfide rich peptide having 6 cysteine residues that can form 3 disulfide bonds (Cys3-Cys20, Cys10-Cys22 and Cys16-Cys28). Six hydrogen bond interactions of McTI with bovine trypsin were observed in molecular docking whereas, additional hydrogen bond interactions were noticed in molecular dynamic simulation studies. In cytotoxicity analysis against MDA-MB-231 cells, crude seed proteins and F12 exhibited a significant dose dependent response with an IC 50 values of 82.10 ± 6.46 µg/ml and 81.13 ± 4.26, respectively. These findings suggest Momordica charantia proteins as a possible anticancer agent. Furthermore, McTI is a valuable addition in the squash inhibitor family.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"52 72 1","pages":"S28"},"PeriodicalIF":0.0,"publicationDate":"2020-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80420063","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}
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.
本专栏重点介绍本出版物的读者感兴趣的最近发表的文章。我们鼓励ABRF成员将他们认为重要和有用的文章信息转发给Clive Slaughter, MCG-UGA医疗合作伙伴，1425 Prince Ave.， Athens GA 30606, USA。电话:(706)713-2216;传真:(706)713-2221;电子邮件;cslaught@uga.edu，或任何编委会成员。文章摘要反映的是审稿人的意见，而不一定是协会的意见。
{"title":"Article Watch: March, 2020.","authors":"","doi":"10.7171/jbt.20-3101-005","DOIUrl":"https://doi.org/10.7171/jbt.20-3101-005","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":"1 1","pages":"36-43"},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84555476","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}
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.
本专栏重点介绍本出版物的读者感兴趣的最近发表的文章。我们鼓励ABRF成员将他们认为重要和有用的文章信息转发给Clive Slaughter, MCG-UGA医疗合作伙伴，1425 Prince Ave.， Athens, GA 30606, USA。电话:(706)713-2216;传真:(706)713-2221;电子邮件:cslaught@uga.edu，或任何编委会成员。文章摘要反映的是审稿人的意见，而不一定是协会的意见。
{"title":"Article Watch: December 2019.","authors":"C. Slaughter","doi":"10.7171/jbt.19-3004-003","DOIUrl":"https://doi.org/10.7171/jbt.19-3004-003","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":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89383020","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}
Nagesh Kishan Panchal, A. Bhale, Radhika Chowdary, V. Verma, S. S. Beevi
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.
{"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}
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.
{"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}
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