DOCSLIB.ORG

Biochemistry 26 Recombinant DNA Technology

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Biochemistry 26 Recombinant DNA Technology . 1 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology Description of Module Subject Name Biochemistry Dr. Vijaya Khader Dr. MC Varadaraj Paper Name 12 Biochemical Techniques Module Name/Title 26 Recombinant DNA Technology 2 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology 1. Objectives Understanding the elements and tools of recombinant technology Various steps in Recombinant DNA technology Expression of prokaryotic gene 2. Concept Map 3 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology 3. Description 3.1 Genetic engineering and Recombinant DNA technology The manipulation of genes isolated from an organism to alter some specific characteristics attributed by the gene is what is generally referred to as genetic engineering. Recombinant DNA technology is a component of genetic engineering. It refers to the process of cutting a known or a target DNA sequence from one organism and introducing it further into another organism thereby altering the genotype of the recipient organism. Genetic engineering finds tremendous applications in fields of medicine, agriculture, and industry. 3.2 What is a Recombinant DNA Recombinant DNA or rec DNA, is a form of artificial DNA that is made through the combination of one or more DNA strands or insertion of one portion of a strand into another, therefore leading to combination of DNA sequences within different species. 3.3 Elements of recombinant DNA technology Listed below are some of the key elements required for recombinant technology: (A) Enzymes: a. Restriction Endonucleases b. Exonucleases c. DNA ligases d. DNA polymerase e. Other enzymes (B) Cloning Vector (C) Host organism (D) DNA insert or foreign DNA (E) Linker and adaptor sequences. 4 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology 3.3.1 Enzymes A number of enzymes are used in recombinant DNA technology. They are briefly described below: (a) Restriction Endonuclease: These enzymes cut DNA molecules at specific sites. The cuts (cleavage) in DNA strands are made only within or near some specific sites called recognition sites/ recognition sequences/ restriction sites. Recognition sites are specific for each restriction enzyme. There are 3 main categories of restriction endonuclease enzymes, namely Type-I, Type-II and Type-III Restriction Endonucleases (Table 1). Table 1 Different types of restriction endonucleases Type-I Restriction Type-II Restriction Type-III Restriction Endonucleases Endonucleases Endonucleases Properties These endonucleases These enzymes are most These are not used for cleave only one strand stable. gene cloning. of DNA. They show cleavage only They are the These enzymes have at specific sites intermediate enzymes the recognition They produce the DNA between Type-I and sequences of about 15 fragments of a defined Type-II restriction bp length length. endonuclease. These enzymes show cleavage in both the strands of DNA, immediately outside then- recognition sequences. Only Type II Restriction Endonucleases are used for gene cloning due to their suitability. Cleavage pattern DNA cleavage takes The recognition They cleave the DNA at place about 1000 bp sequences for Type-II well-defined sites in the away from the 5′ end of Restriction Endonuclease immediate vicinity of the sequence ‘TCA’, enzymes are in the form recognition sequences, 5 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology which is located within of palindromic sequences e.g. Hinf III, etc. the recognition site with rotational symmetry Requirement of Mg They require Mg2+ ions They require Mg2+ ions They require Mg2+ ions 2+ for their functioning for their functioning for cleavage Requirement of ATP They require ATP for They don’t require ATP They require ATP for their functioning for cleavage cleavage Examples EcoK, EcoB Hinfl, EcoRI, PvuII, Alul, Haelll Nature of cleavage by Restriction Endonucleases: Restriction endonuclease can cut the DNA molecule in two ways: 1. They can cleave the two DNA strands at two different points. Such cuts are termed as staggered cuts and this results into the generation of protruding ends or sticky ends. Such ends can pair readily with each other upon being provided with suitable pairing conditions. Another feature of the restriction endonucleases producing such sticky ends is that two or more of such enzymes with different recognition sequences may generate the same sticky ends. 2. They can cleave both strands of DNA simply at the same point within the recognition sequence. Thus, DNA fragments with blunt ends are generated. PvuII, Haelll, Alul are the examples of restriction endonucleases producing blunt ends. Figure 1 below shows the nature of cleavage by restriction enzymes to produce blunt and sticky ends. 6 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology Figure 1 Cleavage by restriction endonucleases: sticky and blunt ends (b) Exonucleases: Exonuclease, as the name suggests, is an enzyme that removes/cuts nucleotides from the ends of a nucleic acid molecule. The enzyme removes nucleotide from the 5′ or 3′ end of a DNA molecule. Unlike endonucleases, these never produces internal cuts within DNA molecule. Examples of some exonucleases used in recombinant DNA technology are Exonuclease Bal31, E. coli exonuclease III, Lambda exonuclease, etc Figure 2 Functioning of exonucleases and endonucleases 7 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology (c) DNA ligase The function of these enzymes is to ligate or join two fragments of DNA. This is achieved by the synthesis of the phosphodiester bond. In recombinant DNA technology, DNA ligases are used for sealing nicks between adjacent nucleotides. (d) DNA polymerases: DNA polymerases are associated with the synthesis of a new complementary DNA strand of an existing DNA or RNA template. Examples of some DNA polymerases used commonly in genetic engineering are: DNA polymerase 1 prepared from E coli. Taq DNA polymerase used in PCR (Polymerase Chain Reaction). Reverse transcriptase that uses RNA as a template for synthesizing a new DNA strand (called as cDNA/complementary DNA). Its main use is in the formation of cDNA libraries. (e) Other enzymes Other enzymes which are also used in rec DNA methods are shown in Table 2. Table 2 Other enzymes used in recombinant DNA technology Enzyme Function Terminal deoxynucleotidyl Adds single stranded sequences to 3′-terminus of the DNA molecule. transferase One or more deoxynbonucleotides (dATP, dGTP, dl IP, dCTP) are added onto the 3′-end of the blunt-ended fragments Alkaline Phosphatase Removes phosphate group from the 5′-end of a DNA molecule Polynucleotide Kinase Adds phosphate group to the 5′-end of a DNA molecule 3.3.2 Cloning Vectors: Cloning vector is a DNA molecule that has the capability to replicate within the host organism. The target DNA is introduced into this vector to produce the recombinant DNA molecule. A great variety of cloning vectors are in use with E. coli which acts as host organism. Sometimes it becomes desirable to use different host for cloning experiments. So, various cloning vectors have been developed based on other bacteria like Bacillus, Pseudomonas, Agrobacterium, etc. and on different eukaryotic organisms like yeast and other fungi. Different types of DNA molecules may be used as cloning vehicles such as they may be plasmids, bacteriophages, cosmids, phasmids or artificial chromosomes 8 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology Cloning The process of introducing of a foreign gene (DNA fragment, isolated from a donor organism) into another (vector) is called cloning. Plasmids Plasmids are circular double stranded DNA molecules, found mostly inside E.coli. Plasmids are also available commercially. 3.3.3 Host Organism: A host organism is important because it facilitates the replication of the recombinant DNA molecule. Most widely used host for recombinant DNA technology is E. coli. This is because cloning and isolation of DNA inserts is very easy in this host. A good host organism should be easy to transform and in which the replication of recombinant DNA is easier. 3.3.4 DNA Insert or Foreign DNA: The desired DNA segment which is to be cloned is called as DNA insert or foreign DNA or target DNA. It can be of viral, plant, animal or bacterial origin. 3.3.5 Linker and Adaptor Sequences: Linkers and adaptors are basically DNA molecules which are used to help in the modifications of cut ends of DNA fragments. These can be joined to the cut ends and hence produce modifications as desired. Both are short, chemically synthesized, double stranded DNA sequences. Linkers have (within them) one or more target sites for the action of one or more restriction enzymes. They can be ligated to the blunt ends of foreign DNA or vector DNA. Then they undergo a treatment with a specific restriction endonuclease to produce cohesive ends of DNA fragments. EcoRI-linker is a common example of frequently used linkers (Figure 3). 9 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology Figure 3 Linker sequences and their role in recombinant DNA technology Adaptors are molecules which have been synthesized chemically and designed to possess one or both sticky ends. The foreign DNA is ligated with adaptor on both ends. This new molecule, so formed, is then phosphorylated at the 5′-terminii and then integrated into the vector DNA to form the recombinant DNA molecule (Figure 4). Figure 4 Adapter sequences and their role in recombinant DNA technology 10 Biochemical Techniques Biochemistry 26 Recombinant DNA Technology 3.4 Steps in Recombinant DNA technology: Expression of prokaryotic gene Step1: Identification and isolation of gene/DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically by restriction endonuclease. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA. The target gene to be cloned can be obtained from various sources such as genomic library, cDNA library, chemical synthesis of gene or using techniques of gene amplification such as PCR Step 2: Selection of a suitable cloning vector. Most commonly used vectors are plasmids and bacteriophages.
Load more

Recommended publications
  • From Genes to Genomes
    From Genes to Genomes Third Edition Concepts and Applications of DNA Technology Jeremy W. Dale, Malcolm von Schantz and Nick Plant University of Surrey, UK ©WILEY-BLACKWELL A John Wiley & Sons, Ltd., Publication Preface xiii 1 From Genes to Genomes 1 1.1 Introduction 1 1.2 Bask molecular biology 4 1.2.1 The DNA backbone 4 1.2.2 The base pairs 6 1.2.3 RNA structure 10 1.2.4 Nucleic acid synthesis 11 1.2.5 Coiling and supercoilin 11 1.3 What is a gene? 13 1.4 Information flow: gene expression 15 1.4.1 Transcription 16 1.4.2 Translation 19 1.5 Gene structure and organisation 20 1.5.1 Operons 20 1.5.2 Exons and introns 21 1.6 Refinements of the model 22 2 How to Clone a Gene 25 2.1 What is cloning? 25 2.2 Overview of the procedures 26 2.3 Extraction and purification of nucleic acids 29 2.3.1 Breaking up cells and tissues 29 2.3.2 Alkaline denaturation 31 2.3.3 Column purification 31 2.4 Detection and quantitation of nucleic acids 32 2.5 Gel electrophoresis 33 2.5.1 Analytical gel electrophoresis 33 2.5.2 Preparative gel electrophoresis 36 vi CONTENTS 2.6 Restriction endonudeases 36 2.6.1 Specificity 37 2.6.2 Sticky and blunt ends 40 2.7 Ligation 42 2.7.1 Optimising Ligation conditions 44 2.7.2 Preventing unwanted Ligation: alkaline phosphatase and double digests 46 2.7.3 Other ways of joining DNA fragments 48 2.8 Modification of restriction fragment ends 49 2.8.1 Linkers and adaptors 50 2.8.2 Homopolymer tailing 52 2.9 Plasmid vectors 53 2.9.1 Plasmid replication 54 2.9.2 Cloning sites 55 2.9.3 Selectable markers 57 2.9.4 Insertional inactivation
    [Show full text]
  • Thermodynamics of DNA Binding and Break Repair by the Pol I DNA
    Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2011 Thermodynamics of DNA binding and break repair by the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus Yanling Yang Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Yang, Yanling, "Thermodynamics of DNA binding and break repair by the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus" (2011). LSU Doctoral Dissertations. 3092. https://digitalcommons.lsu.edu/gradschool_dissertations/3092 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THERMODYNAMICS OF DNA BINDING AND BREAK REPAIR BY THE POL I DNA POLYMERASES FROM ESCHERICHIA COLI AND THERMUS AQUATICUS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Yanling Yang B.S., Qufu Normal University, 2001 M.S., Institute of Microbiology, Chinese Academy Sciences, 2004 M.S. in statistics, Louisiana State University, 2009 December 2011 ACKNOWLEDGMENTS I would like to express my sincere gratitude to some of the people without whose support and guidance none of this work would have been possible. I deeply thank my advisor, Dr. Vince J. LiCata. His guidance and suggestions have been critical to the completion of this research work and the writing of this dissertation.
    [Show full text]
  • Cloning of Gene Coding Glyceraldehyde-3-Phosphate Dehydrogenase Using Puc18 Vector
    Available online a t www.pelagiaresearchlibrary.com Pelagia Research Library European Journal of Experimental Biology, 2015, 5(3):52-57 ISSN: 2248 –9215 CODEN (USA): EJEBAU Cloning of gene coding glyceraldehyde-3-phosphate dehydrogenase using puc18 vector Manoj Kumar Dooda, Akhilesh Kushwaha *, Aquib Hasan and Manish Kushwaha Institute of Transgene Life Sciences, Lucknow (U.P), India _____________________________________________________________________________________________ ABSTRACT The term recombinant DNA technology, DNA cloning, molecular cloning, or gene cloning all refers to the same process. Gene cloning is a set of experimental methods in molecular biology and useful in many areas of research and for biomedical applications. It is the production of exact copies (clones) of a particular gene or DNA sequence using genetic engineering techniques. cDNA is synthesized by using template RNA isolated from blood sample (human). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. Amplify the gene (GAPDH) using primer forward and reverse with the sequence of 5’-TGATGACATCAAGAAGGTGGTGAA-3’ and 5’-TCCTTGGAGGCCATGTGGGCCAT- 3’.pUC18 high copy cloning vector for replication in E. coli, suitable for “blue-white screening” technique and cleaved with the help of SmaI restriction enzyme. Modern cloning vectors include selectable markers (most frequently antibiotic resistant marker) that allow only cells in which the vector but necessarily the insert has been transfected to grow. Additionally the cloning vectors may contain color selection markers which provide blue/white screening (i.e. alpha complementation) on X- Gal and IPTG containing medium. Keywords: RNA isolation; TRIzol method; Gene cloning; Blue/white screening; Agarose gel electrophoresis.
    [Show full text]
  • An Interplay Between Nonsense-Mediated Decay and DNA Damage Response Pathways
    An interplay between nonsense-mediated decay and DNA damage response pathways Fatemeh Ghasemi Master Thesis Department of Biosciences Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO June 2020 © Fatemeh Ghasemi June 2020 An interplay between nonsense-mediated decay and DNA damage response pathways Supervisor: Rafal Ciosk Co-supervisors: Pooja Kumari, Yanwu Guo http://www.duo.uio.no/ Trykk: Reprosentralen, Universitetet i Oslo II Acknowledgement The work presented in this master thesis was carried out at the Department of Biosciences, University of Oslo in the period between April 2019 to June 2020. First and foremost, I’d like to thank my supervisor Rafal Ciosk for giving me the opportunity to work in his group. Thank you for all your help and positivity. I greatly appreciate everything I learned in my time here. Second, I’d like to express my deep gratitude to my co-supervisor Pooja Kumari, without whom I couldn’t have done this. Thank you for your daily guidance and support in the lab, I truly appreciate all the advice you’ve given me. Further, I’d like to thank everyone else in the Ciosk group, especially Yanwu Guo, for all their practical help in the lab, and for writing this thesis. Your input and advice have been greatly appreciated. Divya and Melanie, thank you for cheering me up every single day. Thank you also to all my friends in the Falnes group who helped me out when I was wandering the hallway looking lost. Lastly, I would like to thank my parents for their unending love and support, and for believing in me.
    [Show full text]
  • Recombinant DNA Technology and Its Applications: a Review S
    International Journal of MediPharm Research ISSN:2395-423X www.medipharmsai.com Vol.04, No.02, pp 79-88, 2018 Recombinant DNA Technology and its Applications: A Review S. A. Shinde*, S. A. Chavhan, S. B.Sapkal, V. N. Shrikhande Dr. Rajendra Gode College of Pharmacy, Malkapur Dist- Buldana(MS), India Mob. No:-09890251512 Abstract: Biotechnology which is synonymous with genetic engineering or recombinant DNA (rDNA) is an industrial process that uses the scientific research on DNA for practical applications. rDNA is a form of artificial DNA that is made through the combination or insertion of one or more DNA strands,It offered new opportunities for innovations to produce a wide range of therapeutic products with immediate effect in the medical genetics and biomedicine by modifying microorganisms, animals, and plants to yield medically useful substances.Recombinant DNA technology is playing a vital role in improving health conditions by developing new vaccines and pharmaceuticals. This review gives brief introduction to rDNA and its applications in various fields. Key words: Chimeric DNA, restriction enzymes, Transgenic Plants, Gene Therapy. Introduction: Human life is greatly affected by three factors: deficiency of food, health problems, and environmental issues. Food and health are basic human requirements beside a clean and safe environment. With increasing world's population at a greater rate, human requirements for food are rapidly increasing. Humans require safe- food at reasonable price. Several human related health issues across the globe cause large number of deaths. Approximately 36 million people die each year from noncommunicable and communicable diseases, such as cardiovascular diseases, cancer, diabetes, AIDS/HIV, tuberculosis, malaria.
    [Show full text]
  • A Scientific Update
    Fall/Winter 2017 expressions a scientific update ® ! No witchcraft required in this issue – Clone with Confidence 2 Overcoming the challenges of applying target enrichment for translational research 5 Now available: the NEBNext Direct® BRCA1/BRCA2 Panel 6 NEBNext® Ultra™ II RNA Library Prep kits – Streamlined workflows for lower input amounts 8 No witchcraft required: Special Prices on all Restriction Enzymes and more! 14 Get free tube opener with NEB competent cells 16 New to Molecular Biology? Get your free NEB Starter Pack be INSPIRED drive DISCOVERY stay GENUINE FEATURE ARTICLE Overcoming the challenges of applying target enrichment for translational research by Andrew Barry, M.S., New England Biolabs, Inc. Target enrichment is used to describe a variety of strategies to selectively isolate specific genomic regions of interest for sequencing analysis. The wide array of approaches presents challenges in selecting the appropriate technology for the growing number of research and clinical applications to which the sequencing data will ultimately be applied. INTRODUCTION strates the practical need for continued use of Examples of these hybrid approaches include In recent years, several techniques have emerged target enrichment strategies across the gamut of multiplex extension ligation (3), molecular to enrich for specific genes of interest. When translational research activities. inversion probes (MIPS)/padlock probes (4), determining the appropriate target enrichment nested patch PCR (5), and selector probes (6). technology to use, one must first consider the TARGET ENRICHMENT These technologies can be broadly characterized primary goal of the study. For example, different APPROACHES as having more complex workflows, requiring approaches will be used if the aim is to identify splitting of samples into separate reactions, and known variants already shown to have clinical There are a number of different target enrich- creating challenges in target coverage uniformity.
    [Show full text]
  • GENETICS Chapter 15
    GENETICS Chapter 15 Memorise Understand Importance * Define: phenotype, genotype, gene, locus, * Importance of meiosis; compare/contrast with High level: 10% of GAMSAT Biology allele (single and multiple) mitosis * Homo/heterozygosity, wild type, * Segregation of genes, assortment, linkage, questions released by ACER are related to recessiveness, complete/co-dominance recombination content in this chapter (in our estimation). * Incomplete dominance, gene pool * Single/double crossovers * Note that approximately 75% of the * Sex-linked characteristics, sex determination * Hardy-Weinberg Principle questions in GAMSAT Biology are related * Test cross: back cross, concepts of parental, to just 7 chapters: 1, 2, 3, 4, 7, 12, and 15. F1 and F2 generations; evaluating a pedigree Introduction Genetics is the study of heredity and variation in organisms. The observations of Gregor Mendel in the mid- nineteenth century gave birth to the science which would reveal the physical basis for his conclusions – DNA – about 100 years later. This is the last of the 3 ‘high-level importance’ chapters. Approximately 45% of GAMSAT Biology questions among ACER’s official practice materials contain content related to 3 chapters: 1, 7 and 15, this chapter. Also, be forewarned: despite the fact that a dihybrid cross (BIO 15.3) is time consuming, it appears in 3 separate ACER GAMSAT practice booklets. Multimedia Resources at GAMSAT-Prep.com Open Discussion Boards Foundational Online Videos Flashcards Special Guest THE BIOLOGICAL SCIENCES BIO-267 15.1 Background Information Genetics is a branch of biology which environment. Consider a heterozygote that deals with the principles and mechanics of expressed one gene (dark hair) but not the heredity; in other words, the means by which other (light hair).
    [Show full text]
  • Molecular Cloning and Confirmation of MSH2, an Essential Gene of Human, Involved in DNA Mismatch Repair
    International Journal of Entomology Research www.entomologyjournals.com ISSN: 2455-4758 Received: 16-04-2021, Accepted: 05-05-2021, Published: 21-05-2021 Volume 6, Issue 3, 2021, Page No. 47-53 Molecular cloning and confirmation of MSH2, an essential gene of human, involved in DNA mismatch repair Sarita Kumari*, C Rajesh Department of Biotechnology, Sri Guru Granth Sahib University, Fatehgarh Sahib, Punjab, India Abstract Mismatch repair (MMR) is one such pathway that repair mismatches generated during DNA replication. Errors in MMR cause certain types of cancer including hereditary non-polyposis colorectal cancer, genome instability, abnormalities in meiosis, sterility in mammalian systems and resistance to certain chemotherapeutic agents. In eukaryotes, MSH2 (MutS homologue 2) is a major player in MMR, acting in combination with MSH3 or MSH6 as a heterodimer. MSH2 has been involved in a variety of processes which protect the integrity of the genome. In this study hMSH2 gene was cloned to identifying their role and understanding the functional diversity of protein that will contribute towards the repair mechanisms. Using in frame designed primers through PCR strategy and cloned full-length cDNA fragment hMSH2 gene into pET32b expression vector. The sequence contained an open reading frame of 2805 bp coding for a putative protein of 935 amino acids. Recombinant C-3 clone (pET32b-hMSH2) observed as positive after restriction digestion with expected band size (~8.6kb). This recombinant clone of hMSH2 gene, further used to express the protein and purified protein used to understand the role of mismatch repair protein MSH2. Keywords: DNA damage, DNA repair, mismatch repair (MMR), MSH2 Introduction cause defects in DNA.
    [Show full text]
  • Unit -IX Chapter-11. Biotechnology Principles and Processes
    INDIAN SCHOOL MUSCAT INDIAN SCHOOL MUSCAT INDIAN SCHOOL MUSCAT Questionbank Biology Unit -IX Chapter-11. Biotechnology Principles and processes IMPORTANT POINTS Biotechnology may be defined as the use of microorganisms animals of plants cells of their com- ponents to generate products and services useful to human beings. Genetic engineering and maintenance of sterile condition in chemical engineering process have given the birth to modern biotechonology. The basic principles of Recombinant DNA Technology involve the stages like generation of DNA fragments and selection of the desired pieces of DNA, insertion of the selected DNA into a cloning vector i.e. plasmid, to create a recombinant DNA, Introduction of the recombinant vectors into host cells (e.g. Bacteria), multiplication and reflaction of clones containing the recombinant molecules and expression of gene to produce the desired product. The tools required in the recombinant DNA technology include restriction enzymes,cloning vectors and competent host. The term DNA recombinant technology refer to the transfer of segment of DNA from one organism to another organism (host cell) where it reproduce.The proces involve a sequence of steps like isolation of genetic material,Cutting of DNA at specific site,amplification of gene of interest using PCR,insertion of recombinant DNA into the host cell organism obtaining the foreign gene product and downstream processing. (1)The enzymes that cuts specifically recognition sites in the DNA is known as (a) DNA ligase (b) DNA Polymerase (c) Reverse
    [Show full text]
  • UNIT-I DNA: Definition, Structure & Discovery DNA
    SRINIVASAN ARTS AND SCIENCE COLLEGE (CO-ED) PERAMBALUlR - 621212 DEPARTMENT OF BIOTECHNOLOGY CLASS : I M.SC., BIOTECHNOLOGY SUBJECT: rDNA TECHNOLOGY SUB CODE : P16BT21 UNIT-I DNA: Definition, Structure & Discovery DNA The structure of the DNA double helix. The atoms in the structure are colour-coded by element and the detailed structures of two base pairs are shown in the bottom right. The structure of part of a DNA double helix Deoxyribonucleic acid is a molecule composed of two polynucleotide chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life. The two DNA strands are known as polynucleotides as they are composed of simpler monomeric units called nucleotides. Each nucleotide is composed of one of four nitrogen-containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine [T]), a sugar called deoxyribose, and a phosphate group. The nucleotides are joined to one another in a chain by covalent bonds (known as the phospho-diester linkage) between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The nitrogenous bases of the two separate polynucleotide strands are bound together, according to base pairing rules (A with T and C with G), with hydrogen bonds to make double- stranded DNA. The complementary nitrogenous bases are divided into two groups, pyrimidines and purines.
    [Show full text]
  • Molecular Cloning
    Now includes NEB® Golden Gate Assembly Kit (BsmBI-HF®v2) Molecular Cloning TECHNICAL GUIDE FREE Next Day Delivery www.neb.uk.com OVERVIEW TABLE OF CONTENTS 3 Online Tools 4–5 Cloning Workflow Comparison 6 DNA Assembly Molecular Cloning Overview 6 Overview Molecular cloning refers to the process by which recombinant DNA molecules are 6 Product Selection produced and transformed into a host organism, where they are replicated. A molecular 7 Golden Gate Assembly Kits cloning reaction is usually comprised of two components: 7 Optimization Tips 8 Technical Tips for Optimizing 1. The DNA fragment of interest to be replicated. Golden Gate Assembly Reactions 2. A vector/plasmid backbone that contains all the components for replication in the host. 9 NEBuilder® HiFi DNA Assembly 10 Protocol/Optimization Tips ® DNA of interest, such as a gene, regulatory element(s), operon, etc., is prepared for cloning 10 Gibson Assembly by either excising it out of the source DNA using restriction enzymes, copying it using 11 Cloning & Mutagenesis PCR, or assembling it from individual oligonucleotides. At the same time, a plasmid vector 11 NEB PCR Cloning Kit is prepared in a linear form using restriction enzymes (REs) or Polymerase Chain Reaction 12 Q5® Site-Directed Mutagenesis Kit (PCR). The plasmid is a small, circular piece of DNA that is replicated within the host and 12 Protocols/Optimization Tips exists separately from the host’s chromosomal or genomic DNA. By physically joining the 13–24 DNA Preparation DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest 13 Nucleic Acid Purification becomes part of the new recombinant plasmid and is replicated by the host.
    [Show full text]
  • Substrate-Assisted Catalysis in the Cleavage of DNA by the Ecori and Ecorv Restriction Enzymes
    Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8499-8503, September 1993 Biochemistry Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes (acid base catalysis/mechanism of deavage/nuclease/chemicafly modiffed oligodeoxynucleotide/H-phosphonate) ALBERT JELTSCH, JURGEN ALVES, HEINER WOLFES, GUNTER MAASS, AND ALFRED PINGOUD* Zentrum Biochemie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8, 30623 Hannover, Germany Communicated by Thomas R. Cech, June 14, 1993 ABSTRACT The crystal structure analyses of the EcoRI- stereochemistry of their catalysis make it likely that these DNA and EcoRV-DNA complexes do not provide clear sug- enzymes cleave DNA by a similar mechanism that involves gestions as to which amino acid residues are responsible for the an attack of an activated water molecule in-line with the activation of water to carry out the DNA cleavage. Based on leaving 03' group. Recently, we published (11) a detailed molecular modeling, we have proposed recently that the at- proposal for the mechanism of DNA cleavage by EcoRI and tacking water molecule is activated by the negatively charged EcoRV based on structural data (Brookhaven data bank pro-Rp phosphoryl oxygen of the phosphate group 3' to the entries: 1RlE, 3RVE), molecular modeling, and results of scissile phosphodiester bond. We now present experimental site-directed mutagenesis experiments (ref. 12 and references evidence to support this proposal. (0) Oligodeoxynucleotide therein): Asp-91 and Glu-111 in EcoRI, Asp-74 and Asp-90 in substrates lacking this phosphate group in one strand are EcoRV, and the phosphate at which cleavage occurs were cleaved only in the other strand.
    [Show full text]