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Molecular Biology and History of DNA Sequencing 02-223 Sept. 9 2014 History of DNA Thomas Morgan first James Watson and described Francis Crick proposed Gregor Mendel first linkage and that DNA is a double described patterns of recombination strand with a double inheritance helical structure 1866 1869 1911 1950 1953 Fredrich Edwin Chargaff Miescher first discovered that A and isolated DNA T, and G and C have equal amounts http://www.nature.com/scitable/content/dna-is-a-double-helix-24263 History of DNA Frederick Sanger Arthur Kornberg Hamilton Smith developed dideoxy replicated DNA in- discovered DNA Commercial DNA sequencing ~100 vitro using DNA restriction automated DNA bases/reaction polymerase enzymes synthesizer PCR developed ~1000 bases/ by Kary Mullis reaction 1957 1961 1970 1971 1977 1983 1986 1996 First genome sequenced using Leroy Hood Marshall in-vitro replication by Ray Wu, developed Nirenberg A.D. Kaiser, and Ellen Taylor . automated elucidated the Phage λ, ~5000 nt took over 3 sequencing codons years DNA Polymerase h"p://www.virology.ws/2009/05/10/the-error-prone-ways-of-rna-synthesis/ Even with proofreading, mistakes made every 107-109 Bases 6 Billion Bases in human genome! h"p://www.virology.ws/2009/05/10/the-error-prone-ways-of-rna-synthesis/ Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002. PCR • Polymerase Chain ReacJon • Invented in 1983 • DNA polymerase from Thermus aqua+cus • 2.2x105 error rate Polymerase Chain Reaction (PCR) overview buffer, ssDNA primers, dNTPs, 5’ 3’ DNA polymerase (Taq) DNA sample 3’ 5’ + Mg2+ - enzyme cofactor 5’ 3’ Melt: ~94oC 30 sec Separate DNA strands 3’ 5’ 5’ 3’ Anneal: Tm - 5oC 30 sec 3’ 5’ Hybridize ssDNA primers Extend from primers 5’ 3’ Extend: 72oC 30 sec/kb 3’ 5’ Melt 25-35 Anneal x cycles Extend Let’s perform paper PCR Polymerase Chain Reaction (PCR) overview Starting DNA Final DNA http://www.accessexcellence.org/RC/VL/GG/ polymerase.php Polymerase Chain Reaction (PCR) overview http://www.accessexcellence.org/RC/VL/GG/ polymerase.php http://www.lifetechnologies.com/us/en/home/life-science/ pcr/elevate-pcr-research/pcr-video-library/pcr- animation.html PCR over time h"p://mtbakerBio.com/sites/default/ﬁles/images/RTPCR%20graphSml.gif Sanger Sequencing Following growing DNA strand with ddNTPs All 4 dNTPs added to each. 10% of the following ddNTP added as well ddATP ddGTP ddTTP ddCTP At any base that complements the ddNTP, 10% chance of terminating Paper sequencing Now that we have all these strands of DNA whose final base we know, what do we do with them? Gel Electrophoresis ATGGACCAGTTG ATGGACCAGTT ATGGACCAGT ATGGACCAG ATGGACCA ATGGACC ATGGAC ATGGA ATGG ATG AT A A=green G=yellow T=red C=blue HGP and Celera ABI 3730x (Sanger) Pros and Cons of SS • Polymerase errors • Can only do 1 average out sequence at a time • Long sequences • Need a lot of DNA to (~450 bp) start with • Expensive: 2¢/base Questions? .

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Bacteriophage T7 DNA Polymerase – Sequenase
Bacteriophage T7 DNA polymerase – sequenase The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Zhu, Bin. 2014. “Bacteriophage T7 DNA polymerase – sequenase.” Frontiers in Microbiology 5 (1): 181. doi:10.3389/fmicb.2014.00181. http://dx.doi.org/10.3389/fmicb.2014.00181. Published Version doi:10.3389/fmicb.2014.00181 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12407012 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA REVIEW ARTICLE published: 16 April 2014 doi: 10.3389/fmicb.2014.00181 BacteriophageT7 DNA polymerase – sequenase Bin Zhu* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Edited by: An ideal DNA polymerase for chain-terminating DNA sequencing should possess the Andrew F.Gardner, New England following features: (1) incorporate dideoxy- and other modified nucleotides at an efficiency Biolabs, USA similar to that of the cognate deoxynucleotides; (2) high processivity; (3) high fidelity in Reviewed by: the absence of proofreading/exonuclease activity; and (4) production of clear and uniform Kirk Matthew Schnorr, Novozymes A/S, Denmark signals for detection. The DNA polymerase encoded by bacteriophage T7 is naturally Samir Hamdan, King Abdullah endowed with or can be engineered to have all these characteristics. The chemically or University of Science and Technology, genetically modified enzyme (Sequenase) expedited significantly the development of DNA Saudi Arabia sequencing technology.
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Sequencing.Pdf
Sequencing. Biological sciences has a long and storied history of natural science – identifying, cataloging, and classifying organisms in different environments. Every aspect of biological sciences is being revolutionized by two dominant technologies: cheap and ubiquitous access to DNA sequencing, and cheap and ubiquitous access to high performance computing. Molecular biology was forever altered by the introduction of the polymerase chain reaction (PCR), and that invention, more than any other, has enabled the sequencing revolution. Taxonomy, one of the oldest endeavors of the biologist, is changing from char- acterizing organisms based on morphology to characterizing organisms based on DNA sequences. Revisionist taxonomic approaches are being introduced throughout the tree of life, and no branch on the tree can avoid the sequenc- ing machine. For example, microbial and viral taxonomy are being completely rewritten because of the analysis of DNA sequences (Rohwer & Edwards, 2002; Thompson et al., 2015). All aspects of ecology are being affected by the intro- duction of DNA sequencing: for example, in the marine environment the ecology of everything from the largest (Cammen et al., 2016) to the smallest (Chow & Suttle, 2015; Haggerty & Dinsdale, 2017) organisms is being revised because of sequence analysis. Evolutionary theories about plants and animals are being upended because of the introduction of genome skimming techniques and deep sequencing (Hollingsworth et al., 2016). Sanger Sequencing Two-time nobel Laureate Dr. Fred Sanger developed this sequencing technol- ogy in 1977. The approach uses terminators, dideoxynucleotide triphosphates (ddNTPs), that terminate DNA extension during replication and do not allow the strand to be synthesized further. Thus, every time a ddNTP is added to the growing DNA strand, synthesis stops and the fragement can not be made any longer.
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Sanger Sequencing – a Hands-On Simulation Background And
Sanger sequencing – a hands-on simulation Background and Guidelines for Instructor Jared Young Correspondence concerning this article should be addressed to Jared Young, Mills College 5000 MacArthur Blvd, Oakland, CA 94613 Contact: [email protected] Synopsis This hands-on simulation teaches the Sanger (dideoxy) method of DNA sequencing. In the process of carrying out the exercise, students also confront DNA synthesis, especially as it relates to chemical structure, and the stochastic nature of biological processes. The exercise is designed for an introductory undergraduate genetics course for biology majors. The exercise can be completed in around 90-minutes, which can be broken up into a 50-minute period for the simulation and a follow-up 50-minute (or less) period for discussion. This follow-up could also take place in a Teaching Assistant (TA) led section. The exercise involves interactions between student pairs and the entire class. There is an accompanying student handout with prompts that should be invoked where indicated in these instructions. Introduction Sanger sequencing is an important technique: it revolutionized the field of Genetics and is still in wide use today. Sanger sequencing is a powerful pedagogical tool well-suited for inducing multiple “aha” moments: in achieving a deep understanding of the technique, students gain a better understanding of DNA and nucleotide structure, DNA synthesis, the stochastic nature of biological processes, the utility of visible chemical modifications (in this case, fluorescent dyes), gel electrophoresis, and the connection between a physical molecule and the information it contains. Sanger sequencing is beautiful: a truly elegant method that can bring a deep sense of satisfaction when it is fully understood.
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DNA Sequencing
DNA Sequencing: Sanger Method (Dideoxynucleotide chain termination) Sanger sequencing is a DNA sequencing method in which target DNA is denatured and annealed to an oligonucleotide primer, which is then extended by DNA polymerase using a mixture of deoxynucleotide triphosphates (normal dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs). ddNTPs lack the 3’ OH group to which the next dNTP of the growing DNA chain is added. Without the 3’ OH, no more nucleotides can be added, and DNA polymerase falls off. The resulting newly synthesized DNA chains will be a mixture of lengths, depending on how long the chain was when a ddNTP was randomly incorporated. Manual DNA sequencing example: • First, anneal the primer to the DNA template (must be single stranded): 5’ -GAATGTCCTTTCTCTAAG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5' • Then split the sample into four aliquots including the following nucleotides: "G" tube: All four dNTPs, one of which is radiolabeled, plus ddGTP (low concentration) "A" tube: All four dNTPs, one of which is radiolabeled, plus ddATP "T" tube: All four dNTPs, one of which is radiolabeled, plus ddTTP "C" tube: All four dNTPs, one of which is radiolabeled, plus ddCTP • When a DNA polymerase (e.g. Klenow fragment) is added to the tubes, the synthetic reaction proceeds until, by chance, a dideoxynucleotide is incorporated instead of a deoxynucleotide. This is a "chain termination" event, because there is a 3' H instead of a 3' OH group. Since the synthesized DNA is labeled (classically with 35S-dATP), the products can be detected and distinguished from the template. Note that the higher the concentration of the ddNTP in the reaction, the shorter the products will be, hence, you will get sequence CLOSER to your primer.
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A Beginner's Guide to Next Generation Sequencing
A BEGINNER’S GUIDE TO NEXT GENERATION SEQUENCING youseq.com Next Generation Sequencing Let’s keep things simple. The world of Next Generation Sequencing (NGS) can seem complex and intimidating. It need not be. Let’s start by reminding ourselves what its useful for and why we use it. All of life is coded in it’s DNA. A remarkably simple code of four molecules that act as a blue print to define the proteins that we and all of the organisms we share our planet with are made of. Reading this code is one of the most astonishing achievements that the human species has ever and will ever accomplish. Reading this code helps us to understand how we are made, how we are all related, how errors or mutations in our DNA cause disease and how we may respond best to medicines. It holds the promise to revolutionise healthcare and has already begun to do so. The first human genome “read” was competed in 2001. It took 10 years and the best part of $2.7bn. It was achieved by DNA sequencing. A method by which the sequence of the DNA is read painstakingly in small fragments and then reassembled to create a complete sequence. Sanger Sequencing as it is known, was the method used to achieve the first publication of the first human genome. Next Generation Sequencing is a phrase used to describe a range of technologies that speed up and reduce the cost of DNA sequencing vs the traditional Sanger sequencing. What are all these different Next Generation Sequencing Technologies? Well there are quite a few of them.
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Discovery and Characterization Of
Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 pyrosequencing Margarita López-Uribe, Christine Santiago, Steve Bogdanowicz, Bryan Danforth To cite this version: Margarita López-Uribe, Christine Santiago, Steve Bogdanowicz, Bryan Danforth. Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 py- rosequencing. Apidologie, Springer Verlag, 2013, 44 (2), pp.163-172. 10.1007/s13592-012-0168-3. hal-01201284 HAL Id: hal-01201284 https://hal.archives-ouvertes.fr/hal-01201284 Submitted on 17 Sep 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2013) 44:163–172 Original article * INRA, DIB and Springer-Verlag France, 2012 DOI: 10.1007/s13592-012-0168-3 Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 pyrosequencing 1 1 2 Margarita M. LÓPEZ-URIBE , Christine K. SANTIAGO , Steve M. BOGDANOWICZ , 1 Bryan N. DANFORTH 1Department of Entomology, Cornell University, Ithaca, NY 14853, USA 2Evolutionary Genetics Core Facilities, Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA Received 5 June 2012 – Revised 8 August 2012 – Accepted 18 September 2012 Abstract – The recent implementation of next-generation sequencing for the discovery of microsatellite markers has made this technology the most effective method for generating genetic markers in non-model organisms.
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DNA Polymerases Drive DNA Sequencing-By-Synthesis Technologies: Both Past and Present
View metadata, citation and similar papers at core.ac.uk brought to you by CORE REVIEW ARTICLEprovided by Frontiers - Publisher Connector published: 24 June 2014 doi: 10.3389/fmicb.2014.00305 DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present Cheng-Yao Chen* Protein Engineering Group, Illumina, San Diego, CA, USA Edited by: Next-generation sequencing (NGS) technologies have revolutionized modern biological and Andrew F.Gardner, New England biomedical research. The engines responsible for this innovation are DNA polymerases; Biolabs, USA they catalyze the biochemical reaction for deriving template sequence information. In fact, Reviewed by: DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Suleyman Yildirim, Walter Reed Army Institute of Research, USA Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized Andreas Marx, University of in Sanger’s dideoxy chain-terminating DNA sequencing chemistry. From these humble Konstanz, Germany beginnings followed an explosion of organism-specific, genome sequence information *Correspondence: accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic Cheng-Yao Chen, Protein Engineering bacteria/archaea were modified and tested in today’s standard capillary electrophoresis Group, Illumina, 5200 Illumina Way, San Diego, CA 92122, USA (CE) and NGS sequencing platforms.These enzymes were selected for their efficient incor- e-mail: [email protected] poration of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage φ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate- labeled nucleoside polyphosphates.
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Discovery of Single-Nucleotide Polymorphisms (Snps) in the Uncharacterized Genome of the Ascomycete Ophiognomonia Clavigignenti-Juglandacearum from 454 Sequence Data
Molecular Ecology Resources (2011) 11, 693–702 doi: 10.1111/j.1755-0998.2011.02998.x Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data K.D.BRODERS,*K.E.WOESTE,†P.J.SANMIGUEL,‡ R. P. WESTERMAN‡ and G. J. BOLAND* *School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1, †USDA Forest Service Hardwood Tree Improvement and Regeneration Center, Department of Forestry and Natural Resources, Purdue University, 715 W. State Street, West Lafayette, IN 47907, USA, ‡Department of Horticulture & Landscape Architecture, Purdue University, 625 Agriculture Mall Drive, West Lafayette, IN 47907, USA Abstract The beneﬁts from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One appli- cation of this new sequencing technology is the rapid generation of genomic information to identify putative single- nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217 000 reads, 80% of which collapsed into 16 125 754 bases in 30 339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche’s GS Mapper.
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An Introduction to Next-Generation Sequencing for Cardiology Table of Contents
An Introduction to Next-Generation Sequencing for Cardiology www.illumina.com/technology/next-generation-sequencing.ilmn Table of Contents Part I. Welcome to Next-Generation Sequencing 3 NGS in Clinical Cardiology Research 3 Part II. What is Next-Generation Sequencing? 3 The Basic NGS Workflow 3 Multiplexing 5 Part III. NGS Applications 6 Whole-Genome Sequencing 6 Exome Sequencing 6 Targeted Sequencing 6 Part IV. Summary 6 Glossary 7 References 8 Part I. Welcome to Next-Generation Sequencing NGS in Clinical Cardiology Research In 1990, the beta cardiac myosin heavy chain gene (MYH7) was first implicated in hypertrophic cardiomyopathy (HCM). Since then, many genes have been associated with channelopathies, cardiomyopathies, and aortopathies, and investigators continue to look to the human genome for answers. Researchers can achieve 60% detection of causative variants for certain cardiac conditions (e.g., LQTS, HCM, ACM/ARVC, CPVT) and genetic testing may reveal whether family members are carriers or at high risk for sudden cardiac arrest.1 Recent advances in technology are removing many of the barriers that have traditionally made genetic testing difficult. The value of genetic testing is gaining recognition among leading organizations. For example, in 2011 the Heart Rhythm Society (HRS) and the European Heart Rhythm Association (EHRA) published guidelines on the state of genetic testing for the channelopathies and cardiomyopathies.1 The introduction of next-generation sequencing (NGS) technology has transformed the way scientists and clinical researchers think about genetic information. NGS offers several advantages over existing methods, such as PCR and Sanger sequencing. Because NGS can assess multiple genes in a single assay, it eliminates the need for multiple single-gene tests to identify the causative variant or variants.
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APPLICATION NOTE GeneArt genome editing Using Sanger sequencing to facilitate CRISPR- and TALEN-mediated genome editing workflows In this application note, we show: template is not provided, the break produced by the enzyme is repaired by an error-prone nonhomologous end • Sanger sequencing by capillary electrophoresis can be joining (NHEJ) process. This results in a heterogeneous used to determine the efficiency of genome editing in population of cells with different insertions or deletions primary transformed cultures (indels) around the user-defined break. This process can • Sanger sequencing is an efficient method to confirm be exploited to generate cell lines with a specific gene successful genome edits in transformed cultures, as well knocked out. Alternatively, if a repair sequence is provided, as screen secondary clones for successful editing events the sequence around the break can be repaired using the repair sequence as a template. By selecting the sequence • Applied Biosystems™ Minor Variant Finder Software can for the repair template, precise sequence changes can be be used to determine the frequency of SNP changes in introduced at a user-defined locus within the genome. clones isolated from secondary cultures However, to obtain a clonal population with a homologous Introduction genome edit, several clones from the primary transformed Ever since the double-helical structure of DNA was pool of cells need to be screened. This necessitates elucidated, researchers have developed techniques for two rounds of screening. First, a primary screen must manipulating DNA sequences. However, directing precise be performed to determine the relative fraction of cells sequence changes at user-defined sites has remained a containing an edit.
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