Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics

Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi

• Editore: CRC Press
• Anno: 2022
• ISBN:
  9781000550986
• Edizione: 1
• Argomento: Medicina
• Categoria: Biochimica in medicina

This book covers a range of topics on exploiting Nigeria's mega biodiversity for food security and health; DNA forensic science and its applications; medical biotechnology and biopharmaceutics; medicinal and underutilized plants; impact and mitigation of antibiotic resistance; bioinformatics applications; medical insect biotechnology; etc. The book will be useful reference material for the scientists and researchers working in the fields of nutraceuticals, molecular diagnostics and DNA forensics, biopharmaceuticals and medical biotechnology, nanotechnology, antimicrobials from indigenous plant species, bioinformatics, etc.

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• Emphasizes recent advances in biotechnologies that will help in tackling emerging global health challenges

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• Provides detailed information on how to harness indigenous bioresources including microorganisms and plants for healthcare delivery

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• Introduces new frontiers in the areas of molecular diagnostics and DNA forensic science and bioinformatics with case studies, recent advances in medical insect biotechnology and molecular genetics of pest use towards the exploitation of arthropod midgut components to develop interventions against infectious diseases

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• Reviews bioactive molecules derived from commonly used and underutilized medicinal plants that could be used to develop novel drugs for improved healthcare delivery

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• Discusses current approaches in medical and biopharmaceutical biotechnology, deployment of inexpensive genomics-based vector surveillance for effective disease outbreak prediction and control of mosquito-borne viruses

Hajiya Mairo Inuwa, Ph.D., is Professor in the Department of Biochemistry and Formerly Director, Centre for Biotechnology Research and Training (CBR&T), Ahmadu Bello University, Zaria, Nigeria.

Ifeoma Maureen Ezeonu, Ph.D., is Professor of Medical Microbiology and Molecular Genetics in the Department of Microbiology, University of Nigeria, Nsukka, Nigeria.

Charles Oluwaseun Adetunji, Ph.D., is Associate Professor of Microbiology and Biotechnology and Director of Intellectual Property and Technology Transfer, Edo State University, Uzairue, Nigeria.

Abubakar Gidado, Ph.D., is Professor of Biochemistry and Director of North-East Zonal Biotechnology Centre of Excellence at the University of Maiduguri.

Emmanuel Olufemi Ekundayo, Ph.D., is Associate Professor of Medical Microbiology and Microbial Genetics, Michael Okpara University of Agriculture, Umudike, Nigeria.

Abdulrazak B. Ibrahim, Ph.D., is a Capacity Development Expert at the Forum for Agricultural Research in Africa (FARA) and Associate Professor of Biochemistry, Ahmadu Bello University, Zaria, Nigeria.

Benjamin Ewa Ubi, Ph.D., is a Professor of Plant Breeding and Biotechnology and Director, Biotechnology Research and Development Centre, Ebonyi State University, Abakaliki, Nigeria.

Part 1 Working Group 04 Molecular Diagnostics and DNA Forensics

Handling Editor: Prof. Ifeoma M. Ezeonu

1 Principles and Techniques for Deoxyribonucleic Acid (DNA) Manipulation

Nwadiuto (Diuto) Esiobu

Florida Atlantic University

Ifeoma M. Ezeonu

University of Nigeria

Francisca Nwaokorie

University of Lagos

DOI: 10.1201/9781003178903-2

Contents

1. 1.1 Introduction
2. 1.2 Molecular Structure of the Nucleic Acids
   1. 1.2.1 Chemical Nature of the Nucleic Acids
   2. 1.2.2 The DNA Double Helix
   3. 1.2.3 DNA Renatures as well as Denatures
   4. 1.2.4 Functions of DNA and RNA
3. 1.3 DNA Replication and Gene Expression in Prokaryotes and Eukaryotes
   1. 1.3.1 DNA Replication
   2. 1.3.2 Stages of DNA Replication
      1. 1.3.2.1 Initiation
      2. 1.3.2.2 Elongation
      3. 1.3.2.3 Termination
   3. 1.3.3 Gene Expression
      1. 1.3.3.1 Transcription
      2. 1.3.3.2 Translation (Protein Synthesis)
   4. 1.3.4 Regulation of Gene Expression
      1. 1.3.4.1 Regulation of Gene Expression in Eukaryotes
      2. 1.3.4.2 Regulation of Gene Expression in Prokaryotes
4. 1.4 Laboratory Techniques for DNA Extraction from Various Cell Types
   1. 1.4.1 DNA Isolation, Purification and Quantification
      1. 1.4.1.1 DNA Extraction Steps
      2. 1.4.1.2 Measuring DNA Concentration and Purity
   2. 1.4.2 Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis (PAGE)
      1. 1.4.2.1 Polyacrylamide Gel Electrophoresis Principle
   3. 1.4.3 Other DNA-Based Molecular Techniques
      1. 1.4.3.1 Denatured Gradient Gel Electrophoresis
      2. 1.4.3.2 Recombinant DNA Technologies
      3. 1.4.3.3 Polymerase Chain Reaction – Types and Applications
      4. 1.4.3.4 Restriction Fragment Length Polymorphism (RFLP) and Ribotyping
      5. 1.4.3.5 Sequencing – Sanger and Next Gen Applications
5. 1.5 Conclusion and Recommendations
6. References

1.1 Introduction

There are only two types of cells that constitute the three domains of life on planet earth – Prokaryotic and Eukaryotic cells, with prokaryotes constituting two (Archaea and Bacteria) of the three domains. The notion of Kingdoms has gradually faded away in light of the current understanding of the mechanistic molecular details of transcription and translation processes of cells. The domains of life are distinguished among others on the sequence of the ribosomal ribonucleic acid (rRNA) molecule which is often used as the chronometer gene. At any given point in time cells (unlike acellular entities such as viruses, viroids and prions) contain three major macromolecules that in turn control the synthesis and types of other cellular components. These include deoxyribonucleic acid (DNA), RNA and proteins. The DNA is a sophisticated double-stranded helix whose antiparallel strands are held together by hydrogen bonds. It is a polymer of nucleotides with pentose phosphate backbones linked to nitrogenous bases. The DNA in all cells is made up of four nitrogenous bases namely the Adenine and Guanine (the Purines) and Thymine and Cytosine (the Pyrimidines). The backbone of the DNA consists of the deoxy-pentose sugar and the phosphate group. This backbone has little or no role in the genetic function and coding of the DNA. It can be peeled off and replaced without changing the function of the DNA molecule (Esiobu, 2006), confirming that genetic traits are determined by the nitrogenous base sequence. The complementary base pairing of the molecule and the stereochemistry of its bases allow for high fidelity in replication and regulation of gene expression. The RNA is a polymer of ribonucleotides joined by phosphodiester bonds as in the DNA. They are, however, different from the DNA in three ways: they are single stranded, contain uracil base instead of thymine and contain hydroxyl groups in carbon number 2 of the ribose sugar backbone. The “Central Dogma of Life” describes the established flow of genetic information from the DNA to RNA and then to proteins in extant cells. DNA is the blueprint through which all of the traits and aggregate properties of a microbial cells, plants and animals (morphology, physiology, metabolism, yield, disease resistance, etc.) are governed. Expression of the DNA results in various types of RNA which in turn are translated at the ribosome into proteins.

Seeing the critical importance of the DNA in cellular structure and function, this chapter is dedicated to understanding the molecular structure of the macromolecules. It describes structures in detail, replication mechanisms and presents the fundamental techniques used in studying and manipulating the DNA of cells. A thorough understanding of the blueprint of life is indispensable to carrying out any reasonable biotechnological projects – in silico or in wet labs.

1.2 Molecular Structure of the Nucleic acids

1.2.1 Chemical Nature of the Nucleic Acids

Once DNA was confirmed as the molecule carrying the genetic information of cells, the next challenge that immediately faced geneticists was determination of its structure.

Two types of nucleic acids are found within cells, DNA and RNA. By the 1940s, a fair amount of information was already available about the nucleic acids. It was known that nucleic acids are large polymers composed of nucleotide subunits. Each nucleotide consists of three composite molecular groups: a central 5-carbon atom sugar, connected to a phosphate group and a single-ringed or double-ringed nitrogenous base (Figure 1.1a).

FIGURE 1.1a Molecular structure of a nucleotide. (Source: Wikipedia.)

FIGURE 1.1b The chemical structures of the sugar moieties in RNA and DNA. (Source: Wikipedia.)

FIGURE 1.1c Structure of DNA bases. Adenine and Guanine are the purine bases, Cytosine and thymine are the pyrimidine bases. (Source: https://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/nucacids.htm.)

FIGURE 1.1d Structure of the pyrimidine base, Uracil; found only in RNA. (Source: Wikipedia.)

FIGURE 1.1e Basic Nucleotide Structure, showing the base, sugar and phosphate groups. In a deoxyribonucleotide, the C-1 carbon atom of a deoxyribose is bonded to N-1 of a pyrimidine (a) or N-9 of a purine (b). The sugar and base of a nucleotide are together referred to as a nucleoside. The addition of a phosphate makes it a nucleotide (c). (Source: Wikipedia.)

FIGURE 1.1f Formation of a phosphodiester linkage between two nucleotides. (Source: https://gfycat.com/discover/nitrogenous-base-gifs.)

FIGURE 1.1g Watson-Crick Model of DNA.

FIGURE 1.1h The structure of DNA showing the complementary and antiparallel arrangement. Note that one chain ends in 5′ hydroxyl group while the other ends in a 3′ hydroxyl. (Source: https://www.google.com/search?q=antiparallel+arrangement+of+dna&client.)

The sugar moiety differs between DNA and RNA; the exact difference being in carbon-2. In the structure depicted above, the 5-carbon sugar is ribose, with a hydroxyl group at carbon-2. This makes the nucleotide a ribonucleotide and the nucleic acid, RNA. In DNA, on the other hand, the sugar is deoxygenated at carbon-2 (deoxyribose) as shown in Figure 1.1b, making the nucleotide a deoxyribonucleotide and the nucleic acid, DNA.

The nitrogenous base in a nucleotide can be either a purine (double-ringed) or a pyrimidine (single-ringed). Four types of nitrogenous bases are found in DNA: adenine (A), guanine (G), cytosine (C) and thymine (T). Structures of these bases are shown in Figure 1.1c. RNA, on the other hand, contains the bases A, G, C, but T is replaced by another pyrimidine, uracil (U). The structure of uracil is shown in Figure 1.1d.

In the nucleotide structure, the central sugar moiety is connected to the nitrogenous base by a bond between its C-1 carbon atom and the N-1 atom of a pyrimidine or the N-9 atom of a purine as shown in Figure 1.1e. The sugar and base of a nucleotide are together referred to as a nucleoside. The addition of a phosphate makes it a nucleotide. Both DNA and RNA are therefore polynucleotides, because they are polymers of nucleotide subunits. Free unincorporated nucleotides exist as triphosphates, with three phosphate groups, but during incorporation into the nucleic acid chain, only one phosphate group is incorporated, while two phosphates are released as pyrophosphate (PPi).

In DNA and RNA chains, nucleotide subunits are connected into long chains by covalent linkages formed between the phosphate group of one nucleotide and a hydroxyl group on another nucleotide. Such bonds are called phosphodiester linkages. In a growing polynucleotide chain, the hydroxyl group (OH) on carbon-3 of the sugar of an existing nucleotide serves as the acceptor molecule, to which the phosphate group on carbon-5 of the incoming nucleotide attaches (Figure 1.1f).

Because the linking of nucleotides is always from the carbon-3 OH group (the 3′ end), the growths of both DNA and RNA are directional, strictly in the 5′ to 3′ direction; the oldest end being the 5′ end, while the youngest end is the 3′ end. This also makes the 3′-OH group an extremely important functional group in a polynucleotide, as without it, the nucleotide chain would cease to grow. This property has been exploited in some DNA technologies, such as Sanger dideoxy or chain terminating sequencing technique.

Although the chemical composition of DNA was already known by the 1940s, an understanding of how its atoms were linked together and the establishment of their three-dimensional arrangements in space was necessary to learn how the information in DNA is transmitted through cell generations (Watson et...