DNA Sequencing

5 Key excerpts on "DNA Sequencing"

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  eBook - ePub

  Molecular Biology and Biotechnology

  • Ralph Rapley, Ralph Rapley(Authors)
  • 2021(Publication Date)
  • Royal Society of Chemistry

    (Publisher)

  10 Genome Sequencing

  Ioly Kotta-Loizou

  *a

  a 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’.

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  10.2 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.

  2

  Simultaneously, 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

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  eBook - ePub

  Bioinformatics

  A Practical Handbook of Next Generation Sequencing and Its Applications

  • Lloyd Low, Martti Tammi(Authors)
  • 2017(Publication Date)
  • WSPC

    (Publisher)

  Chapter 1

  Introduction to Next Generation Sequencing Technologies

  Lloyd Lowa and Martti T. Tammib

  a Perdana University Centre for Bioinformatics (PU-CBi), Block B and D1, MAEPS Building, MARDI Complex, Jalan MAEPS Perdana, 43400 Serdang, Selangor, Malaysia. b Biotechnology & Breeding Department, Sime Darby Plantation R&D Centre, Selangor, 43400, Malaysia.

  A Brief History of DNA Sequencing

  In 1962 James Watson, Francis Crick and Maurice Wilkins jointly received the Nobel Prize in Physiology/Medicine for their discoveries of the structure of deoxyribonucleic acid (DNA) and its significance for information transfer in living material.1 The secret of DNA in orchestrating living activities lies in the arrangement of the four bases (i.e. adenine, thymine, guanine and cytosine). The linear sequence of the four bases can be considered as the language of life with each word specified by a codon that is made up of three bases. It was an interesting puzzle to figure out how codons specify amino acids. In 1968, Robert W. Holley, HarGobind Khorana and Marshall W. Nirenberg were awarded the Nobel Prize in Physiology/Medicine for solving the genetic code puzzle. Now it is known that collection of codons direct what, where, when and how much proteins should be made. Since the discovery of the structure of DNA and the genetic code, deciphering the meaning of DNA sequences has been an ongoing quest by many scientists to understand the intricacies of life.

  The ability to read a DNA sequence is a prerequisite to decipher its meaning. Not surprisingly then, there has been intense competition to develop better tools to sequence DNA. In the 1970s, the first revolution in DNA Sequencing technology began and there were two major competitors in this area. One was the commonly known Sanger sequencing method

  2 ,3

  and another was the Maxam–Gilbert sequencing method.4

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  eBook - ePub

  Genomics and Clinical Diagnostics

  • David Whitehouse, Ralph Rapley(Authors)
  • 2019(Publication Date)
  • Royal Society of Chemistry

    (Publisher)

  10 However, the field of DNA Sequencing has been revolutionised by so called next-generation sequencing (NGS) or second-generation sequencing (SGS), with methodology such as Illumina sequencing by synthesis, among others. Indeed more recently third-generation sequencing methods, such as Nanopore sequencing, have also and continue to be developed, providing many exciting alternatives for DNA sequence analysis.

  3.2.1 Sanger ‘Chain Termination’ Sequencing

  Sanger sequencing is an enzymatic method that requires single-stranded DNA as the template. Traditionally this demanded that the DNA fragment of interest be inserted and cloned into the specialised bacteriophage vector termed M13, which is naturally single-stranded. Although M13 is still available for use, the advent of PCR has provided the means not only to amplify a region of any genome or cDNA for which primer sequences are available, but also to very quickly generate the corresponding nucleotide sequence. This has led to an explosion in the accumulation of DNA sequence information and has provided much impetus for many areas of genomic analysis.

  The Sanger method is simple and elegant and in many ways mimics the natural ability of DNA polymerase to extend a growing nucleotide chain based on an existing template. Initially the DNA to be sequenced is allowed to hybridise with an oligonucleotide primer, which is complementary to a sequence adjacent to the 3′ side of DNA. The oligonucleotide will then act as a primer for synthesis of a strand of DNA, catalysed by DNA polymerase. Either one of the deoxynucleotide triphosphates (dNTPs) required for DNA synthesis is labelled (or all four in some methods) with a fluorescent molecule or, alternatively, the primer may be labelled to provide the means of detection following the completion of the method.

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  eBook - ePub

  Practical Bioinformatics

  • Michael Agostino(Author)
  • 2012(Publication Date)
  • Garland Science

    (Publisher)

  CHAPTER 1

  Introduction to Bioinformatics and Sequence Analysis

  Key concepts •  The scope of bioinformatics •  The origins and growth of DNA databasess •  Evidence of evolution from bioinformatics •  Example sequence analysis and displays using human Factor IX

  1.1  INTRODUCTION

  We are witnessing a revolution in biomedical research. Although it has been clear for decades that exploring the genetics of biological systems was crucial to understanding them, it was far too expensive and complex to consider obtaining genetic sequences for that exploration. But now, acquiring genetic sequences is affordable and simple, and data are being generated at unprecedented rates. The heart of understanding all this sequence lies in bioinformatics sequence analysis, and this book serves as an introduction to this powerful study of DNA, RNA, and protein sequence.

  Bioinformatics concerns the generation, visualization, analysis, storage, and retrieval of large quantities of biological information. The generation of biomedical data, including DNA sequence, in its raw form does not involve bioinformatics skills. But in order for that sequence to be usable, it must be analyzed, annotated, and reformatted to be suitable for databases. These are all bioinformatics activities. Many of these activities can be automated, but their development and support come from someone with skills or experience in bioinformatics.

  Once the data have been made available, how do you analyze the data? Is there text like DNA and protein sequence files? If yes, it should be presented in a way to allow interpretation or easy input into programs for analysis. Or is there so much information that data are represented graphically? This form of data reduction is quite powerful and without it we would be staring at pages and pages of sequence without, literally, seeing the big picture.

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  eBook - ePub

  Bionanotechnology

  Principles and Applications

  • Anil Kumar Anal(Author)
  • 2018(Publication Date)
  • CRC Press

    (Publisher)

  Stryjewska et al. (2013) For growth hormone deficiency Human growth hormone For anemia treatment Erythropoietin For prevention of blood clotting Activase For the development of live-attenuated vaccines Tetravalent dengue (DEN) vaccine Lee et al. (2012) 3.6    DNA Sequencing

  DNA molecule is composed of deoxyribose sugar, phosphate group, and four different types of nitrogenous bases, namely, cytosine, adenine, guanine, and thymine. DNA Sequencing, therefore, is the study of the arrangement of these nucleotides in a strand of DNA molecules, which regulates the blueprint of life and provides genetic and biological information at the molecular level. DNA Sequencing involves sequencing of genomic DNA, complementary DNA (cDNA), and analysis of epigenetic modification of genomic DNA molecules (Mitsui et al. 2015). With the advancement in molecular biology, DNA Sequencing technique also experiences improvement, and the DNA Sequencing methods are classified into four generations.

  Sanger’s chain termination or dideoxy first generation technique involves utilization of DNA polymerase, single-stranded DNA template, primer DNA, chemical analogs of the deoxyribonucleotides (dNTPs), and dideoxynucleotides (ddNTPs). The molecule ddNTPs lack 3′hydroxyl, which is needed to bond with 5′ phosphate of the next dNTP molecule, thus leading to the termination of extension. Briefly, the process starts with annealing of primer to specific region of template DNA, which starts to synthesize DNA strand in the presence of DNA polymerase. During DNA extension reaction, addition of radiolabeled ddNTPs at fraction of the concentration of standard dNTPs results in random incorporation of ddNTPs in DNA strand terminating the extension. The experiment is conducted in four tubes each with specific amount of the four dNTPs and the resulting fragments with same 5′-end, whereas 3′-end is determined by specific ddNTP, which is run on four lanes of polyacrylamide gel. Nucleotide sequence in the original template is determined by autoradiography, which produces a radioactive band in the corresponding lane (Heather and Chain 2016).

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