google-site-verification=ZLcPltAq6Squr3Y1qHa0pGUzUhEJ5pyLCd_ZMn9Uh-o

Today is Friday, May 3, 2024

Molecular Genetics

This page features selected topics especially helpful in basic laboratory techniques and DNA-based sample identification. The publications, presentations, and web links have varying target audiences; some are informative to the layman while others may assist professionals in the field. We hope you find this page useful!

Practical Lab Tips: In March 2004, Greg was invited to speak to a group of graduating Biology majors about scientific careers. Practical Lab Tips emerged as a simple handout for students ready to face the “real world.” Greg considers himself the scribe of this document since the true authors are life experiences. It was such a useful guide that he made some slight modifications so it applies to both novice and veteran scientists. In the subsequent years, portions (especially the PCR section) were incorporated into countless instructions, publications, methods, and SOPs.

Binomial Nomenclature: The correct format for the genus species or a species’ scientific name.

BenchFly: Do you need a refresher on how to perform colony PCR? Do you even know what “colony PCR” is? Learn about it and other protocols in action at www.benchfly.com. Check out this site for short, how-to videos regarding basic molecular biology tips and techniques on biological, chemical, proteonomic, instrumental, and other methods.

Reverse Complement: Here is simple and convenient online tool to compute the reverse complement of DNA sequences.

Amplicon to Copy Number: Need help converting the nanograms of amplicon into copy numbers? This website from IDT provides the formula you need. Click here to access an on-line calculator; all you’ll need is the nanograms of DNA and the sequence length!

DNA Learning Center: Molecular genetics concepts made easy! Watch 3D animations, set-your-own-pace tutorials, presentations, and more about cell functions. Find out how DNA and and RNA work inside a living cell. Find out how molecular biology research is conducted through videos of different laboratory techniques.

DNA Replication Animations: Watch this presentation by Drew Berry as he discusses DNA replication in real time from double helix to karyokinesis.

STRBase: Short Tandem Repeat DNA Internet DataBase, National Institute of Standards & Technology

One option to analyze your raw STR data is by using Osiris (Open Source Independent Review and Interpretation System), available for free through NCBI.

A handy site on forensics algorithms is Forensic Mathematics of DNA Matching.

Cellosaurus: Visit Cellosaurus to get the details on over 60,000 cell lines studied in biomedical research. Click here for a great summary of this project.

Cellosaurus’ STR Similarity Search Tool, developed by Amos Bairoch and Thibault Robin, is one of the most powerful and comprehensive STR databases available for cell line research. Greg Sykes worked with the developers during the beta testing stage. This STR search tool is best used in the Google Chrome browser.

International Cell Line Authentication Committee (ICLAC): An organization formed by an independent group of scientists to promote cell line authentication and alert researchers of known misidentified cell lines.

List of Prokaryotic Names with Standing in Nomenclature (LPSN): Quite simply, this list provides all of the bacterial species known to science, including synonyms and taxanomic revision history. TIP: Within the list, click on the ¤ symbol to open a page containing more information.

DNA Barcoding: We are all familiar with Universal Product Codes (UPC)—those barred labels on nearly everything you buy. All the clerk needs to do is scan it with a laser, the computer searches for the price for that item, and the sale rings up on the register. What if we could do the same thing for life, except having a species name come up instead of a price? Turns out we can! Using semi-conservative genes, scientists can identify many different organisms. The target specimen is generally identified (e.g., animal, fungus, bacteria, archaebacteria, etc.), DNA is purified from a small tissue sample, and that DNA is analyzed on a platform based on its general grouping. For example, the animal specimens have the cytochrome c oxidase I (CO1) gene analyzed, bacteria need the 16S gene read, and so forth.

In CO1 Barcoding, the resulting data are compared to voucher specimen DNA sequences. The videos and tutorials on these sites can teach you more about the actual DNA sequencing steps:

Vouchers are specimens whose species were independently verified by experts using non-DNA characteristics (e.g., morphology, biochemical analysis, etc.) to be an accurate species representative. Only sequence data with high Quality Scores (QualScores) are entered as vouchers. The ever-expanding voucher databases are huge, covering many species; current major database developments include fish, insect, and bird identification. This technology finds diverse applications: collected field species identification, law enforcement and the illegal wildlife/animal parts trade, seafood verification, and cell culture identification. For an overview on how CO1 Barcoding works, please click here.

Explore these links for more specific information and various data analysis:

  • Consortium for the Barcodes of Life: Identifying animal species using Barcode DNA; provides an overview of different DNA barcoding initiatives, projects, and resources
  • Barcode of Life Data (BOLD) Systems: Analyze your barcode data here; emphasis is on animal data using the CO1 gene, but expanding into the Fungi and possibly other taxonomic kingdoms
  • NCBI Basic Local Alignment Search Tool (BLAST): Houses many different sequences—a good site to run a preliminary analysis, however some sequence data are unverified, may be incorrectly labeled, are derived from low QualScore data, and/or do not come from voucher specimens. Other handy NCBI pages:
  • Ribosomal Database Project (RDP): Using a curated database, performs search analysis of both your Bacterial and Archaeal 16S DNA sequence data. NOTE: Due to funding problems, the RDP website is offline until further notice.
    • For classic (not DNA based) methods of identifying bacterial species, use MicrobeLibrary

Need help troubleshooting your Sanger sequencing experiments? Try the Biomolecular Core Facility’s Sequencing Guidelines or the Centre for Genomic Sciences’ Troubleshooting Guide.

Molecular Biology Papers Authored by Greg Sykes

American Type Culture Collection Standards Development Organization Workgroup ASN-0002-2022 (Christopher T. Korch, Erin M. Hall, Wilhelm G. Dirks, Gregory R. Sykes, Amanda Capes-Davis, Tanya Barrett, John M. Butler, Richard M. Neve Raymond W. Nims, Douglas R. Storts, Fang Tian, Roland M. Nardone). 2022. Human Cell Line Authentication: Standardization of Short Tandem Repeat (STR) Profiling. ANSI/ATCC ASN-0002-2022. Copyrighted by ATCC and the American National Standards Institute (ANSI). Manassas, VA, USA. https://webstore.ansi.org/standards/atcc/ansiatccasn00022022. Hard copies are available at https://www.atcc.org/products/135-ansi-str.

American Type Culture Collection Standards Development Organization Workgroup ASN-0003 (Almeida J, Alston-Roberts C, Bauer B, Burnett E, Cole K, Cooper J, Cooper JK, de Mars M, Elmore E, Furtado M, Hanner R, Kohara A, Korch C, Nims R, Oien N, Reid Y, Schindel, DE, Schlottmann S, Sohrabi A, Stoeckle M, Sykes G). 2015. Species-Level Identification of Animal Cells through Mitochondrial Cytochrome c Oxidase Subunit 1 (CO1) DNA Barcodes. ANSI/ATCC ASN-0003-2015. Copyrighted by ATCC and the American National Standards Institute (ANSI). Manassas, VA, USA.
http://webstore.ansi.org/RecordDetail.aspx?sku=ANSI%2fATCC+ASN-0003-2015 Hard copies are available at https://www.atcc.org/products/135-ansi-str.

Capes-Davis, Amanda, Yvonne A. Reid, Margaret C. Kline, Douglas R. Storts, Ethan Strauss, Wilhelm G. Dirks, Hans G. Drexler, Roderick A. F. MacLeod, Gregory Sykes, Arihiro Kohara, Yukio Nakamura, Eugene Elmore, Raymond W. Nims, Christine Alston-Roberts, Rita Barallon, Georgyi V. Los, Roland M. Nardone, Paul J. Price, Anton Steuer, Jim Thomson, John R. W. Masters, Liz Kerrigan. 2012. Match criteria for human cell line authentication: Where do we draw the line? International Journal of Cancer. http://onlinelibrary.wiley.com/doi/10.1002/ijc.27931/abstract

American Type Culture Collection Standards Development Organization Workgroup ASN-0002 (Alston-Roberts C, Barallon R, Bauer SR, Butler J, Capes-Davis A, Dirks WG, Elmore E, Furtado M, Kerrigan L, Kline MC, Kohara A, Los GV, MacLeod RAF, Masters JR, Nardone M, Nims RW, Price PJ, Reid YA, Shewale J, Steuer AF, Storts DR, Sykes G, Taraporewala Z, Thomson J). 2011. Authentication of Human Cell Lines: Standardization of STR Profiling. ANSI/ATCC ASN-0002-2011. Copyrighted by ATCC and the American National Standards Institute (ANSI). Manassas, VA, USA.
http://webstore.ansi.org/RecordDetail.aspx?sku=ANSI%2fATCC+ASN-0002-2011

American Type Culture Collection Standards Development Organization Workgroup ASN-0002 (Alston-Roberts C, Barallon R, Bauer SR, Butler J, Capes-Davis A, Dirks WG, Elmore E, Furtado M, Kerrigan L, Kline MC, Kohara A, Los GV, MacLeod RAF, Masters JR, Nardone M, Nims RW, Price PJ, Reid YA, Shewale J, Steuer AF, Storts DR, Sykes G, Taraporewala Z, Thomson J). 2010 (June). Cell line misidentification: the beginning of the end. Nature Reviews Cancer 10(6):441-448. PubMedID 20448633. http://www.nature.com/nrc/journal/v10/n6/full/nrc2852.html

Barallon, Rita, Steven R. Bauer, John Butler, Amanda Capes-Davis, Wilhelm G. Dirks, Eugene Elmore, Manohar Furtado, Margaret C. Kline, Arihiro Kohara, Georgyi V. Los, Roderick A. F. MacLeod, John R. W. Masters, Mark Nardone, Roland M. Nardone, Raymond W. Nims, Paul J. Price, Yvonne A. Reid, Jaiprakash Shewale, Gregory Sykes, Anton F. Steuer, Douglas R. Storts, Jim Thomson, Zenobia Taraporewala, Christine Alston-Roberts, and Liz Kerrigan. 2010. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues. In Vitro Cellular & Developmental Biology – Animal 46(9):727-732. http://www.springerlink.com/content/u642314114610652/

Nims, Raymond W., Greg Sykes, Karin Cottrill, Pranvera Ikonomi, and Eugene Elmore. 2010. Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification. In Vitro Cellular & Developmental Biology – Animal 46(10):811–819. http://www.springerlink.com/content/g5v4987247722115/

Cooper, Jason, Greg Sykes, Steve King, Karin Cottrill, Natalia V. Ivanova, Robert Hanner, and Pranvera Ikonomi. 2007. Species identification in cell culture: a two-pronged molecular approach. In Vitro Cellular & Developmental Biology – Animal 43(10):344-351. http://www.springerlink.com/content/rp8142p07444t134/

Josephson, R., G. Sykes, Y. Liu, C. Ording, W. Xu, X. Zeng, S. Shin, J. Loring, A. Maitra, M.S. Rao, and J.M. Auerbach. 2006. A molecular scheme for improved characterization of human embryonic stem cell lines. BioMed Central (BMC) Biology 4:28. http://www.biomedcentral.com/1741-7007/4/28

Durkin, A. Scott, Edward Cedrone, Gregory Sykes, Debra Boles, and Yvonne A. Reid. 2000. Utility of gender determination in cell line identity. In Vitro Cellular & Developmental Biology – Animal 36(6):344-347. http://www.springerlink.com/content/d6g74333w8129gq3/

Sykes, G.R., A.H. Christensen, and P.M. Peterson. 1997. A chloroplast DNA analysis of Chaboissaea (Poaceae:Eragostideae). Systematic Botany 22(2):291-302. http://www.jstor.org/discover/10.2307/2419458?uid=3739936&uid=2133&uid=2&uid=70&uid=4&uid=3739256&sid=21101490840711

Symposium and Poster Presentations

Sykes, Greg. 2016. Mitochondria-Targeting PCR and CO1 Barcode Sequence Analyses as Alternatives to Isoenzymology. International Society for BioProcess Technology (ISBioTech) 4th Fall Meeting. [View]

Sykes, Greg. 2015. Mitochondrial Multiplex and CO1 Barcode Analyses as Alternatives to Isoenzymology. International Society for BioProcess Technology (ISBioTech) 3rd Fall Meeting.

Sykes, Greg. 2012. Cell Culture Species Identification through Molecular Genetics. International Society for BioProcess Technology (ISBioTech) 2nd Annual Meeting.

Reid, Y.A., G. Sykes, P. Ikonomi, and E. Cedrone. 2003. Authentication of cell lines. Molecular Biology of the Cell 14 (supplement): 477a. Abstract #2671 for poster presentation in the 43rd American Society for Cell Biology Annual Meeting.

Sykes, Greg (speaker), Robyn Hedges, Ming Hui, and Yvonne Reid. 2002. Human cell line quality control through STR analysis. 35th Annual Middle Atlantic Regional Meeting, American Chemical Society, May 28-30, 2002. Abstract #67 (page 56). [View]

Sykes, Greg and Yvonne Reid. 2001. Occurrence and stability of STR microvariant alleles in established human cell lines. Molecular Biology of the Cell 12 (supplement): 523a. Abstract #2879 for poster presentation in the 41st American Society for Cell Biology Annual Meeting. [View]

Return to Science and Nature Homepage.