Biological Sciences
DNA Sequencing
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. This technique allows scientists to understand the genetic makeup of an organism, identify mutations, and study genetic variations. By decoding the sequence of DNA, researchers can gain insights into the functioning of genes and their role in various biological processes.
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10 Key excerpts on "DNA Sequencing"
eBook - ePub- Ralph Rapley(Author)
- 2021(Publication Date)
- Royal Society of Chemistry(Publisher)
10 Genome SequencingIoly Kotta-Loizou*aa Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, UK, * E-mail: [email protected]Next-generation sequencing (NGS) is a rapidly developing technology with a wide range of applications in biological sciences. Historically, DNA Sequencing has evolved from the first to the second and third generation in less than 50 years. This chapter aims to provide guidance for designing NGS studies, performing NGS experiments and analysing NGS data, focusing mostly on differential gene expression analysis.
10.1 Introduction
DNA Sequencing determines the order of the four bases adenine (A), cytosine (C), guanine (G) and thymine (T) in a segment of DNA. The genomic DNA sequence encodes initially the intermediate RNA sequence and finally the translated protein sequence (central dogma of molecular biology). The protein sequence determines initially the protein structure and finally the protein function (Anfinsen's dogma). Therefore, knowledge of the DNA where the genetic information is stored provides insight into the proteins synthesised, the macromolecules responsible for the majority of functions within the living organisms and viruses. This knowledge is a prerequisite for genetic engineering, i.e. directed modification of the DNA sequence in order to create proteins with new functions, and essential in a range of theoretical and applied fields including, but not limited to, medicine, agriculture, livestock, microbiology, biodiversity, evolution and forensics. Nowadays it is believed that ‘in the long view of history, the impact of the DNA Sequencing will be on a par with that of the microscope’.110.2 A Brief History of DNA Sequencing
10.2.1 Early Steps
The first sequencing methods were published in the mid-1970s. Allan Maxam and Walter Gilbert (Harvard University) developed what is known as Maxam–Gilbert sequencing, based on base-specific chemical modification and subsequent partial cleavage of the DNA phosphodiester bonds at the positions of the modified bases.2Simultaneously, Frederick Sanger and his colleagues (Cambridge University) developed what is known as Sanger sequencing, based on the incorporation of chain-terminating dideoxynucleotides (ddNTPs), present at a low ratio compared with deoxynucleotides (dNTPs), during an in vitro DNA synthesis reaction.3 ,4
eBook - PDFDNA Sequencing
Methods and Applications
- Anjana Munshi(Author)
- 2012(Publication Date)
- IntechOpen(Publisher)
Section 1 Methods of DNA Sequencing 1 DNA Representation Bharti Rajendra Kumar B.T. Kumaon Institute of Technology, Dwarahat,Almora, Uttarakhand, India 1. Introduction The term DNA Sequencing refers to methods for determining the order of the nucleotides bases adenine,guanine,cytosine and thymine in a molecule of DNA. The first DNA sequence were obtained by academic researchers,using laboratories methods based on 2- dimensional chromatography in the early 1970s. By the development of dye based sequencing method with automated analysis,DNA Sequencing has become easier and faster. The knowledge of DNA sequences of genes and other parts of the genome of organisms has become indispensable for basic research studying biological processes, as well as in applied fields such as diagnostic or forensic research. DNA is the information store that ultimately dictates the structure of every gene product, delineates every part of the organisms. The order of the bases along DNA contains the complete set of instructions that make up the genetic inheritance. The rapid speed of sequencing attained with modern DNA Sequencing technology has been instrumental in the sequencing of the human genome, in the human genome project. Fig. 1. DNA Sequence Trace DNA can be sequenced by a chemical procedure that breaks a terminally labelled DNA molecule partially at each repetition of a base. The length of the labelled fragments then identify the position of that base. We describe reactions that cleave DNA preferentially at guanines,at adenines,at cytosine and thymines equally, and at cytosine alone. When the product of these four reactions are resolved by size,by electrophoresis on a polyacrylamide gel, the DNA sequences can be read from the pattern of radioactive bands. The technique DNA Sequencing – Methods and Applications 4 will permit sequencing of atleast 100 bases from the point of labelling.
eBook - PDF- Fitch, J. Patrick(Authors)
- 2002(Publication Date)
73 5 DNA Sequencing DNA Sequencing has become a high-throughput process for determining the ordered base pairs in a strand of DNA. Manufacturing techniques, including statistical process control, are now routine. An example throughput metric is the number of DNA bases sequenced per day per dollar. Some sequencing centers report their daily and monthly production online. Commercial organizations such as Incyte Genomics (http://incyte.com) and Celera Genomics (http://celera.com) have significant DNA Sequencing capacity. Public sequencing activities for the Human Genome Project were mostly conducted in five large centers: the Sanger Center, the Washington University Genome Sequencing Center, the Department of Energy/University of California Laboratory Joint Genome Institute, the Whitehead Institute for Biomedical Research at Massachusetts Institute of Technology, and the Baylor College of Medicine. There are several sites on the worldwide web that summarize DNA Sequencing progress. The European Bionformatics Institute Genome Monitoring Table (MOT) page at http://www.ebi.ac.uk/genomes/mot/ is updated daily with sequencing progress for several eukaryotes. The Institute for Genome Research (TIGR) maintains a list of published microbial genomes and chromosomes at http://www.tigr.org/tdb/mdb/mdbcomplete.html. The NCBI also has a list of microbial DNA sequence, including completed microbial genomes from both archaea and bacteria. 5.1 Sequencing Approaches In most DNA Sequencing approaches, the DNA sequence is assembled from many shorter, overlapping subsequences. The sequence of a strand less than a couple of thousand base pairs in length is measured using four-color electrophoresis. Our description of DNA Sequencing here begins with a simplified description of sequencing chemistry, followed by discussions of electrophoresis and computerized base calling and assembly.
eBook - ePub- Julia Lodge, Peter Lund, Steve Minchin(Authors)
- 2007(Publication Date)
- Taylor & Francis(Publisher)
7 Sequencing DNA Learning outcomes: By the end of this chapter you will have an understanding of: the enzymatic sequencing procedure developed by Fredrick Sanger and colleagues examples of modifications to basic enzymatic sequencing methodology, including PCR-based methods and methods for high-throughput sequencing how to interpret both gel-based and chromatogram-based DNA sequences strategies employed for genome sequencing the methods used to sequence the human genome 7.1 Introduction DNA Sequencing is one of the lynch pins of modern molecular biology. When DNA Sequencing techniques were first developed they allowed scientists to sequence short gene-sized segments of the genome of the organism they were studying. The procedure was intimately linked to library screening since the gene was invariably isolated from either a genomic or cDNA library using procedures that have been described in Chapter 5. Once the sequence of the gene was known a clue to the structure and function of the protein product could be gleaned by comparisons with other known genes and prediction of possible structural motifs. Analysis of DNA and protein sequences will be discussed in Chapter 8. From the DNA, sequence hypotheses could be developed and tested. Often this would involve mutagenesis of the gene and, again, sequencing became a vital tool either to confirm that site-directed mutants had been successfully obtained or to screen random mutants to determine their sequence. More recently, DNA Sequencing has been a central part of the “genomic revolution”
eBook - PDF- Brian K. Nunnally(Author)
- 2005(Publication Date)
- CRC Press(Publisher)
157 7 DNA Sequencing for Genome Analysis Jeffrey P. Tomkins, Todd C. Wood, and Dorrie Main CONTENTS Introduction ........................................................................................................... 157 EST Sequencing .................................................................................................... 158 Development of Sequence-Ready Genomic Frameworks .................................... 159 Whole-Genome Sequencing .................................................................................. 163 Conclusion ............................................................................................................. 172 References .............................................................................................................. 173 INTRODUCTION Genome analysis has developed over time through the various fields of genetics, cytogenetics, biophysics, biochemistry, and molecular biology. Each of these disci-plines has contributed to our understanding of the nature of inheritance and how genes contribute toward an organism’s phenotype. We may briefly define a genome as the complete set of DNA instructions for a given organism, organized into chromosomal units and containing the genes which code for the organism’s traits. As a result, historically separate fields of biological study find union within the arena of genomics. Deciphering the genetic code or precise order of nucleotides represents one of the most fundamental steps in genomic analysis. Genome sequenc-ing in its various forms serves as a foundation for analyses of transcription, gene regulation, chromosome structure, genetic pathologies, biochemical pathways, and evolution. There are a number of approaches to genome sequencing that may be taken depending on the size of the genome, its complexity, and the availability of funds.
eBook - PDFSustainable Agriculture
Nanotechnology and Biotechnology for Crop Production and Protection
- Vishnu D. Rajput, Abhishek Singh, Karen Ghazaryan, Tatiana M. Minkina, Abdel Rahman M. Al-Tawaha(Authors)
- 2024(Publication Date)
- De Gruyter(Publisher)
This mini review deals with evolution of NGS tech- nique, its working mechanism and application in the field of agriculture. Keywords: Next-generation sequencing, genotyping by sequencing, molecular ap- proaches, parallel sequencing RNA sequencing 20.1 Introduction Among most evolutionary discoveries in the field of molecular biology is the identifi- cation of DNA as the genetic material. DNA was identified as genetic material by Os- ✶ Corresponding author: Neha Chakrawarti, Department of Genetics and Plant Breeding, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India, e-mail: [email protected] Indra Deo, Department of Genetics and Plant Breeding, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India Rajshree Verma, Department of Plant Pathology, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India https://doi.org/10.1515/9783111234694-020 wald Theodore Avery in 1944. The present structure of DNA as we see was defined by James D. Watson and Francis Crick in 1953. They demonstrated DNA as combination of four nucleotides arranged in double helical strand structure which are complemen- tary and antiparallel. DNA sequences have now become the most versatile research tool in molecular biology. DNA Sequencing technologies could provide wide range of solutions in form of gene identification, gene sequencing, molecular cloning, phyloge- netic studies, comparative and fundamental studies. An ideal DNA Sequencing tech- nology (Fig. 20.1) should be efficient, fast, error-free, cost-effective, and easy to use. The first methods for sequencing DNA using chain termination and fragmentation techniques were created in the 1970s by Sanger and colleagues [1] and Maxam and Gilbert [2]. These sequencing technologies provided tools for complete gene sequenc- ing and complete genome sequencing.
- Leland J. Cseke, Ara Kirakosyan, Peter B. Kaufman, Margaret V. Westfall(Authors)
- 2016(Publication Date)
- CRC Press(Publisher)
141 7 DNA Sequencing and Analysis Leland J. Cseke and Lance Larka DNA.sequencing.is.a.necessary.technique.for.almost.all.molecular.biology.studies.including.DNA. cloning,.characterization,.mutagenesis,.DNA.recombination,.regulation.of.gene.expression, 1–3 .and. protein.expression.There.are.several.well-established.methods.for.nucleic.acid.sequencing,.and.in. recent.years,.advances.in.automated.sequencing.have.made.DNA.sequence.analysis.an.easy.and. routine.process.The.present.chapter.describes.in.detail.the.preparation.of.DNA.for.sequencing 4–6 . DNA.sequencing.by.dideoxynucleotides.chain.termination 2,3 .and.cycle.sequencing 1,4–6 .These.tech-niques.are.the.foundation.of.most.sequencing.applications;.however,.with.the.advent.of.automated. CONTENTS 71. Preparation.of.DNA.for.Sequencing. 142 711. Preparation.of.Double-Stranded.Plasmid.DNA 142 712. Preparation.of.Single-Stranded.Template.DNA. 142 7121. Preparation.of.Single-Stranded.M13.DNA. 142 7122. Preparation.of.Single-Stranded.DNA.Using.Helper.Phage.R408. 143 713. Purification.of.Double-Stranded.Lambda.DNA. 143 714. Preparation.of.Double-Stranded.DNA.Fragments. 144 72. DNA.Sequencing.by.Dideoxynucleotide.Chain.Termination144 73. Dye.Cycle.Sequencing.and.Automated.Sequencing. 145 731. Selection.of.Oligonucleotides. 145 732. Dye.Cycle.Sequencing.and.Automated.Sequencing. 146 733. DNA.Sequencing.Using.an.Automated.Sequencer. 148 7331. Sequencing.Reactions. 148 7332. Sample.Preparation.for.Injection.150 7333. Analysis.of.Sequence.Chromatograms. 151 74. Troubleshooting.Guide. 151 741. Weak.Signals. 151 742. Extensions.Appear.Short.(Read.Length.Limited.to.Less.than.350.Bases). 153 743. Chromatogram.Anomalies. 153 75. Importance.of.Genome.Sequencing. 153 751. Shotgun.Sequencing. 155 752. Next.Generation.Sequencing.Platforms.156 7521. Solexa.Sequencing. 158 7522. Other.Sequencing.Technologies. 158 76. Sequence.Assembly.and.Annotation. 159 761. Assembly.Algorithms.
- Gabor L. Hornyak, H.F. Tibbals, Joydeep Dutta, John J. Moore(Authors)
- 2008(Publication Date)
- CRC Press(Publisher)
The first methods developed to sequence DNA were termination seq-uencing developed by Frederick Sanger and Howard Chadwell and chemical sequencing developed by Allan Maxam and Walter Gilbert, both during the 1970s. Because of the difficulty and labor involved in performing sequencing, only short portions of genomes were sequenced at first. Since the award of the Nobel Prize in 1980 to Sanger and Gilbert, intensive and large-scale efforts have been made to extend these first steps. Worldwide coordinated projects were started to achieve rapid and inexpensive sequencing of complete genomes for any organism or individual, culminating in the first nearly complete draft sequences Biomolecular Nanoscience 791 of the human genome in 2001 and 2002, spurred by the U.S. government-sponsored Human Genome Project [93–98]. The development of genome sequencing was a unique large-scale develop-ment involving many cooperative and competitive efforts, analogous for biology to space exploration programs or to the high-energy physics accelerator projects for discovery of elemental particles. A number of important steps marked the rapid progress in ability to decipher genome sequences. The first sequencing of a complete organism was carried out in 1983 by a team of scientists at the U.K. Medical Research Council for the Epstein–Barr virus, whose genome had appro-ximately 170,000 base pairs (170 kb). The same year Mullis and coworkers devel-oped a way to amplify replication of strands of DNA by repetitive cycling, which could be readily automated. This development, called variously the polymerase chain reaction (PCR), DNA thermocycling, or DNA amplification, made it feasi-ble to obtain workable amounts of DNA from very small amounts of starting material and revolutionized DNA Sequencing and cloning [99]. Initially, DNA Sequencing reactions are only capable of analyzing the sequence of a few thousand nucleotides at a time.
- Amit Kumar Tyagi(Author)
- 2023(Publication Date)
- Wiley-Scrivener(Publisher)
Once more, this is a cutting-edge field of cellular biology where the potential position of activity and inactivity needs to be widely explored. If genomics is the study of the assets of the genome, genetics is the study of how characters or phenotypes are approved down through groups [2]. The identification of genetic differ- ences related to neuropsychiatric disorders and treatment significances has thus amplified self-assurance that these findings will rapidly be functional in the clinic to improve diagnosis, disease risk forecast, and patient reply to drug therapy. Slower DNA segments can be sequenced using the shot- gun method, clone by clone, whole genome, Maxam Gilbert, and Sanger sequencing methods. Sanger chemistry, the “original” sequencing method, reads through a DNA template shaped during DNA synthesis using spe- cially branded nucleotides [3]. The Sanger method is used read 1000 to 1200 base pairs (bp) thanks to a number of practical developments, but it is still imperfect to the 2-kilo base pair (kbps) [4]. In this book chapter focus, the hub of genomics, Genome Sequencing Methods, Variation of Genome Sequencing, Diseases and Disorders, and Future Prospects. 1.2 Hub of Genomics There are slightly more than 20,000 human protein-coding genes. Still, every one of these classifications typically codes for numerous proteins Introduction to Genomics and Genetics 5 thanks to mechanisms like uncommon concerning, differing strand tran- scripts, and others. There may be up to five determined transcripts per gene sequence, giving some confirmation. Two percent of the human genome’s DNA balance contains the real protein-encoding orders. It is now generally acknowledged that tens of thousands of genomic areas encode “noncod- ing RNA transcripts.” These RNAs display a role in controlling messenger RNA translation and gene entrance (mRNAs) [5].
eBook - PDFNanomedicine
Science, Business, and Impact
- Michael Hehenberger(Author)
- 2015(Publication Date)
- Jenny Stanford Publishing(Publisher)
—Edward Teller (1908–2003) The first DNA sequences were obtained in the early 1970s by academic researchers. Early forms of nucleotide sequencing were based on chromatography, laboratory techniques for the separation of mixtures that were invented in 1900 by Michail Tsvet 81 and first applied by him to the extraction of plant pigments such as chlorophyll and carotene. RNA sequencing was done first because it was easier to deal with a single strand of a helical molecule than working with the full double helix. Between 1972 and 1976, Walter Fiers and his coworkers at Ghent (Belgium) were able to sequence the first complete gene and the complete genome of a viral genome, bacteriophage MS2. 82 During the 1970s, Sanger at the Medical Research Council in Cambridge, UK, developed his method of DNA Sequencing with chain-terminating inhibitors, 83 and Walter Gilbert and Allan Maxam at Harvard in Cambridge, Massachusetts, developed DNA Sequencing by chemical degradation. 84 In 1980, Sanger and Gilbert shared one half of the Nobel Prize 85 for their contributions concerning the determination of base sequences in nucleic acids, with Paul Berg of Stanford University, capturing the other half for his fundamental studies of the biochemistry of nucleic acids, with particular regard to rDNA. 3.6.1 Chemical Sequencing Maxam–Gilbert sequencing is based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing , this method’s interesting history is described in detail by Gilbert in his Nobel lecture. 86 The basic idea is to find a repressor protein that can protect a short stretch of 110 Genetics and DNA Sequencing DNA from degradation, caused, for example, by digestive enzymes. Subsequently, they found that protein binding would block not only enzymes but also chemical methylation, the addition of CH 3 groups to the bases in DNA.
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