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Recombinant DNA Technology
Recombinant DNA Technology
By
Siddra Ijaz and Imran Ul Haq
Recombinant DNA Technology By Siddra Ijaz and Imran Ul Haq This book first published 2019 Cambridge Scholars Publishing Lady Stephenson Library, Newcastle upon Tyne, NE6 2PA, UK British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Copyright © 2019 by Siddra Ijaz and Imran Ul Haq All rights for this book reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. ISBN (10): 1-5275-3758-7 ISBN (13): 978-1-5275-3758-3
TABLE OF CONTENTS List of Illustrations ................................................................................... vii List of Tables ............................................................................................. ix Preface ....................................................................................................... xi Abbreviations .......................................................................................... xiii Chapter One ................................................................................................ 1 Gene Cloning: An Overview Chapter Two ............................................................................................. 19 Gene Libraries Construction and Screening Chapter Three ........................................................................................... 25 Concepts and Approaches in Plant Genetic Transformation Chapter Four ............................................................................................. 41 Marker Genes: Tools for Tracking Plant Transformation Events Chapter Five ............................................................................................. 51 Techniques in Molecular Biology Chapter Six ............................................................................................... 67 Genome Editing Approaches Chapter Seven ........................................................................................... 75 Application of Recombinant DNA Technology
Table of Contents
vi
Appendices ............................................................................................... 83 Glossary .................................................................................................... 85 Bibliography ............................................................................................. 89 Index ....................................................................................................... 101
LIST OF ILLUSTRATIONS Figure 1.1: A schematic diagram of gene cloning, representing generation of recombinant DNA molecule and its multiplication Figure 1.2: Cleavage action of restriction endonucleases Figure 1.3: Blue White Selection for selecting the recombinant molecule Figure 2.1: A schematic representation of general procedure for genomic library construction Figure 2.2: An illustrative diagram of general procedure for cDNA library construction Figure 3.1: An illustration of Agrobacterium tumefaciens and Ti plasmid Figure 3.2: Signal cascading for the transfer of T-DNA from Agrobacterium tumefaciens to the plant genome Figure 3.3: A pictorial representation of T-DNA transfer and integration from Agrobacterium tumefaciens to plant genome Figure 3.4: A schematic diagram of illegitimate recombination Figure 3.5: Gene gun: a physical approach for gene delivery into plant genome Figure 4.1: Cre-loxP recombination system Figure 5.1: TaqMan probe based qPCR assay Figure 5.2: Molecular Beacon: A probe of qPCR technique Figure 5.3: Scorpion Probe: A probe of qPCR technique Figure 5.4: A schematic diagram of southern blotting
List of Illustrations
viii
Figure 5.5: A schematic diagram of northern blotting Figure 6.1: Zinc Finger nucleases (ZNFs): Mechanism of action for site directed genome editing Figure 6.2: CRISPR/CAS9 system Figure 7.1: A pictorial depiction of mechanism of action of Bt toxin on insect after feeding on BT crop plant expressing cry protein toxin
LIST OF TABLES Table 1.1: Cloning vectors Table 1.2: List of restriction enzymes along with ends generated
PREFACE The progressing scientific revolution is resulting in changes in the established rules of human-biotic world interactions. Researchers are now able to identify and characterize genes for almost all biological functions, modify and incorporate them into living cells, exchange genetic materials between species and produce clones (genetic blueprints) of all living beings through the art of Recombinant DNA Technology. Genetic engineering has emerged as the ultimate solution to most everyday life problems, from food safety/security to improving health. A plethora of life’s daily issues are now dependent on genetic engineering, either through the development of genetically modified organisms or of new drugs/vaccines to combat diseases. All this is possible due to emerging knowledge in genetic engineering.
This book details different Recombinant DNA Technology techniques and their applications. It is intended to address the approaches of current genetic engineering and their wizardly applications in laboratories, as well as in the field. The book is aimed at professional biologists, university students and scientists in general. This book will have an evocative impact on the minds of its readers and will provide comprehensive knowledge on the further potential of Recombinant DNA Technology. In this book, we have tried to leave no stone unturned to present the complex field of genetic engineering in a fluid way, by using simple and reader-friendly language without compromising its scientific value.
The book is comprised of seven chapters. The first chapter is on Gene Cloning, while the second is about the Construction of Gene Libraries. The third chapter concerns principles and methods for the genetic Transformation of Plants, and light has been shed on Marker Genes and their excision in the fourth chapter. Techniques in Molecular Biology and Approaches in Gene Editing are discussed in the fifth and sixth chapters respectively. The last chapter is about the Applications of Recombinant DNA Technology. We are hopeful that by the end, this effort will lead to the creation of some opportunities to add a little to the already existing ocean of genetic engineering.
Siddra Ijaz, PhD Imran Ul Haq, PhD
ABBREVATIONS A Adenine AdoMet S-adenosyl methionine Amp Ampicillin ATP Adenosine Tri Phosphate attB Bacterial attachment Site attP Phage attachment Site BAC Bacterial Artificial Chromosome bar A gene, confers resistance to bialaphos
herbicide BCIP-T 5-bromo-4-chloro-3-indolyl phosphate,
p-toluidine salt BT Bacillus thuringiensis C Cytosine C2H2 Cys2His2 CaMV35S Cauliflower mosaic virus 35S Cas 9 CRISR-associated protein 9 cDNA Complementary DNA Cre Cyclic recombinase CRISR Clustered regularly interspaced short
palindromic repeats crRNA CRISPR RNA Ct Cycle threshold DNA Deoxyribonucleic acid dNTPs deoxyribonucleotide triphosphate DSBs Double stranded Breaks EDTA Ethylenediamine tetra acetic acid FLP Flippase G Guanine GDS Gel Documentation System GFP Green Fluorescent Protein GMO Genetically Modified Organisms HART Hybrid Arrest Translation HCl Hydrochloric acid HDR Homology Directed Repair He Helium
Abbrevations
xiv
HR Homologous Recombination HRP Horseradish peroxidase HRT Hybrid Release Translation Int Integrase IPTG Isopropyl β-D-1-thiogalactopyranoside IR Illegitimate Recombination ISSR Inter Simple Sequence Repeat KJ kilojoule LoxP Locus of X-over in P1 bacteriophage LSC Large single copy M Molar MCSs Multiple Cloning Sites MgCl2 Magnesium Chloride mRNA Messenger RNA NaCl Sodium Chloride NaOH Sodium Hydroxide NBT Nitro Blue Tetrazolium NHEJ Non Homologous End Joining NLS Nuclear localization sequence NPC Nuclear pore complex ORF Open Reading Frame ori Origin of Replication PAGE Polyacrylamide Gel Electrophoresis PAGE Polyacrylamide Gel Electrophoresis PAM Protospacer adjacent motif PCR Polymerase Chain Reaction PEG Polyethylene Glycol PI domain PAM interacting domain qPCR Quantitative PCR R gene Resistance gene RAMP Repair associated mysterious protein RAPD Radom Amplified Polymorphic DNA RBS Recombinase Binding site Ri plasmid Root inducing plasmid RNA Ribonucleic acid RNA Ribonucleic acid rpm Revolution per minute SDS Sodium dodecyl Sulphate SSC Small single copy SSC Sodium Chloride-Sodium Citrate SSCP Single Strand Conformational Polymorphism
Recombinant DNA Technology
xv
SSR Simple Sequence Repeat ssTDNA single stranded transfer DNA T Thymine TAE Tris acetate EDTA TAL Transcription activator-like TALEN Transcription activator-like effector nucleases Taq polymerase DNA polymerase from Thermus aquaticus T-DNA Transfer DNA Ti plasmid Tumor inducing plasmid tracrRNA transactivating CRISPR RNA UV Ultraviolet Vir gene Virulence gene X-Gal 5-bromo-4-chloro-3-indolyl-β-D-
galactopyranoside YAC Yeast Artificial Chromosome ZFNs Zinc Finger Nucleases
CHAPTER ONE
GENE CLONING: AN OVERVIEW
In gene cloning, a set of experiments to assemble DNA fragments is used to generate a recombinant DNA molecule. Gene cloning is the core aspect of Recombinant DNA Technology. In principle, it involves the joining of DNA fragments from different sources. In this technique, the DNA fragment is ligated with a vector to develop a recombinant DNA molecule. Then this recombinant DNA molecule is delivered into the host cell for multiplication, so as to produce multiple identical copies of this molecule.
Methods for the cloning of a particular segment of DNA into a vector are categorized as either (1) ligase dependent or (2) ligase independent methods. However, ligase dependent methods are generally practiced for cloning. Ligation dependent cloning methods are further subdivided into two groups, (1) cohesive ended ligation and (2) blunt ended ligation. These approaches require restriction enzymes to generate cohesive or sticky ends, and multiple enzymatic modifications for blunt ended ligation. PCR development has made this task quite simple and fast.
PCR amplified fragments are cloned through different methods, such as through engineering restriction enzymes sites at the 5' end of primers, ligase independent cloning and TA cloning. Among all these, TA cloning is the more efficient cloning strategy. The TA vector has a 3'-T overhang, while Taq polymerase has the ability to generate a 3'-A overhang in amplified PCR products, by introducing a single 3'-A into them. These overhangs are complementary to each other, thus facilitating the direct cloning of a PCR product into the vector. Therefore, in this chapter, an overview of the general cloning strategies is briefly presented.
Chapter One
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BASIC FEATURES OF VECTORS
A cloning vector must have the ability to replicate independently in the host cell. Vectors to be used in cloning experiments must have the following features:
They must be a circular DNA molecule. They must have their origin of replication (ori) for their autonomous
replication within a host. They must contain selectable marker gene sequences. They must have multiple cloning sites (MCSs) region.
VECTORS FOR CLONING
Different vectors are used in molecular biology for cloning purposes, e.g. plasmid, λ phage, cosmids, BAC (bacterial artificial chromosome) and YAC (yeast artificial chromosome). They accept different sizes of DNA fragments/inserts to carry them as recombinant. A list of cloning vectors and insert sizes which they may carry is given in the table below (Table 1.1).
Table 1.1: Cloning vectors
BASIC STEPS OF GENE CLONING DNA manipulative and DNA modifying enzymes with diverse properties, as well as a series of experimentations involved in gene cloning, are described in detail below. The general steps involved in gene cloning are also illustrated pictorially in Fig. 1-1.
Vector Insert size
Plasmid ~ 10 Kb
λ phage ~ 23 Kb
Cosmid ~ 45 Kb
BAC ~ 350 Kb
YAC ~ 1000 Kb
Gene Cloning: An Overview
3
Figure 1.1: A schematic diagram of gene cloning, representing generation of recombinant DNA molecule and its multiplication (idea taken from Brown 2006).
Gene isolation:
In the first step, a gene, or any DNA fragment to be used in cloning is isolated using different methods, such as a PCR based on sequence-specific primers, to amplify DNA fragments from extracted genomic DNA, RNA, organellar DNA, gene libraries etc. This step in gene cloning is mediated by polymerases.
Chapter One
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Restriction digestion
Restriction enzymes (molecular scissors) are involved in the restriction digestion step of gene cloning. These restriction enzymes are categorized into two broad classes based on their mechanism of action; (1) Exonucleases (2) Endonucleases. A diverse range of restriction enzymes is available. The vector and DNA fragment must both be cut with the same kind of restriction enzymes for generating compatible ends in order to facilitate their proper joining.
Multiple cloning sites (MCSs) or polylinkers in a vector are the restriction sites that are specific to restriction enzymes (restriction endonucleases). The sites at which these molecular scissors make the cut are known as recognition sites or restriction sites. These sites may be made up of 4, 5, 6 or 8 base pair-long symmetrical inverted repeats (Palindromic) as well as of asymmetrical sequences; hence, based on the sequence length of recognition sites, endonucleases are referred to as either four cutters, five cutters, six cutters or eight cutters. Among them, four cutters and six cutters are the most commonly used molecular scissors in recombinant DNA technology. The restriction enzymes are cut in different ways and thereby may generate sticky ends or staggered ends (5' protruding ends or 5' phosphate overhangs, and 3' protruding ends or 3' hydroxyl overhangs) as well as blunt ends or flush ends (Fig. 1-2). The ligation efficiency of compatible sticky ends is more than that of blunt ended DNA molecules. Thus, to turn blunt ended DNA into a sticky ended molecule, linkers and adapters are used. Adapters are the short stretches of synthetic oligonucleotides with sticky ends, whereas linkers are blunt ended synthetic oligonucleotides but with restriction enzymes sites, which generate sticky ends.
Classification of restriction endonucleases
Restriction endonucleases are generally classified into four broad categories based on sequence specificity, cleavage position, the composition of their subunits and the requirements of their co-factors etc. These are classified as:-
Type I restriction endonucleases Type II restriction endonucleases Type III restriction endonucleases Type IV restriction endonucleases
Gene Cloning: An Overview
5
Figure 1.2: Cleavage action of restriction endonucleases (a) blunt ended DNA molecule (b) 5' protruding sticky ended DNA molecule (c) 3' protruding sticky ended DNA molecule (idea taken from Russell 2009).
Type I Restriction Endonucleases
Type I restriction enzymes are made up of multi-subunits and are complex in nature. They have independent subunits for recognition, cleavage of DNA molecules at specific sites and their modifications. The location of these endonucleases’ recognition sites is far from their cleavage sites, which make these restriction enzymes no longer suitable for biotechnological applications. These restriction enzymes require AdoMet (S-adenosyl methionine), an Mg+ ion and ATP (Adenosine triphosphate) for their activity.
Type II Restriction Endonucleases
Type II restriction enzymes’ recognition sites are located within or close to their restriction sites. This feature gives them specificity and defined positions for cutting. They recognize symmetrical palindromic sequences. This class of restriction endonucleases is of practical value for use in gene cloning and other molecular analyses in laboratories. However, they don’t have any AdoMet and ATP requirements for their activity. A list of restriction enzymes which are being used in recombinant DNA technology is given in the Table 1.2.
Chapter One
6
Type III Restriction Endonucleases
These restriction enzymes make a cleavage at DNA molecules outside their recognition sites. They recognize asymmetrical sequences of inverse orientation like the Type I class of restriction enzymes. This group of enzymes also requires AdoMet and ATP for their functioning.
Type IV Restriction Endonucleases
These restriction endonucleases act on modified methylated DNA molecules as recognition sites.
DNA modification
DNA molecules are modified by the addition or removal of their chemical groups according to the cloning strategy’s needs. Various types of DNA modifying enzymes, such as alkaline phosphatase (the mediating removal of the phosphate group at the 5' terminal of a DNA molecule), polynucleotide kinase (the mediating addition of the phosphate group at the 5' terminal of a DNA molecule) and terminal deoxynucleotidyl transferase (the mediating addition of more than one nucleotide at the 3' terminal of a DNA molecule) are used in gene cloning experimentation.
Ligation reaction Restriction digestion as well as modification (in some cases) of a DNA molecule and vector is followed by a ligation reaction. In ligation reactions, the ligase enzyme (molecular scissors) is used to mediate the joining of the DNA fragment and vector molecule together into a recombinant DNA molecule. A ligation mixture contains,
1) Unligated molecules a) Unligated vectors b) Unligated DNA fragments
2) Self-ligated vector molecules 3) A recombinant DNA molecule
Delivery to host and clone selection
The ligation mixture is delivered into the host cell for the selection of a desired clone (a recombinant DNA molecule), and then to produce multiple copies of this clone. However, the selection of the clone is made using a selective agent (in the culture medium required for the growth of a host cell) against a selectable marker gene present in the vector backbone.
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s Ce
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s Hsp
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s Ita
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s Kas
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s N
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s Cf
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s Cl
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s
Gen
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g: A
n O
verv
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11
Csp4
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Chap
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12
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Tabl
e 1.2
: A L
ist o
f res
tric
tion
enzy
mes
alo
ng w
ith en
ds g
ener
ated
by
thes
e end
onuc
leas
es, u
sed
in re
com
bina
nt D
NA
te
chno
logy
. Mad
e af
ter c
onsu
lting
info
rmat
ion
prov
ided
by
http
://re
base
.neb
.com
/reba
se/re
base
.htm
l
Gene Cloning: An Overview
13
Protocols in Gene Cloning DNA molecules generated from different sources may be used in gene cloning for diverse downstream applications. This section will explain the general cloning workflow and protocols involved in gene cloning experimentation, and describe how the DNA fragment of interest (the insert) is amplified through a Polymerase Chain Reaction (PCR). Restriction sites of specific restriction enzymes are engineered at the 5' ends of specific primers to the DNA fragment of interest. PCR analysis is performed to amplify the DNA fragment of interest flanked by specific sites against particular restriction enzymes. The PCR product (amplicon) is resolved on agarose using gel electrophoresis. Agarose gel is prepared in a 0.5X TAE buffer (Appendix A). The gel is visualized and photographed using the gel documentation system (GDS). Subsequent to this procedure, the following methods are employed.
DNA Elution
For DNA elution, agarose gel containing a DNA fragment of the required size is excised with a clean scalpel. Gel extraction is performed by using available gel extraction kits (according to the manufacturer’s protocol) for eluting the required fragment.
PCR purification
Instead of DNA elution, PCR purification may also be performed if the PCR product is a lone specified amplicon. For purification, PCR purification kits are available. Therefore, PCR purification is performed according to the manufacturer’s protocol which gives instructions on which PCR purification kit to use.
Competent Cell Formation
Competent Escherichia coli (E. coli) cells are prepared so as to build up their ability to take DNA up. For cloning purposes, plasmidless strains of E. coli are used. The general protocol for the preparation of bacterial competent cells is given below.
Take a loop of E. coli fresh culture and transfer into L.B liquid for broth cultures (Appendix B).
Incubate this culture at 37°C @180rpm overnight and then transfer 2ml from overnight culture into 100ml of LB liquid in a flask.
Chapter One
14
Incubate it at 37°C @ 180rpm to get a fresh culture. Centrifugate (for 5 minutes, at 3000-4000 rpm, 4°C) to harvest E.
coli cells. Discard the supernatant. Add 0.1M MgCl2 to pellet Resuspend it by gentle mixing and then centrifugation (5 minutes,
3000-4000 rpm, 4°C). Discard the supernatant. Add 0.1M CaCl2 to pellet. Resuspend it by gently swirling and incubating on ice for 15-30
minutes followed by centrifugation (for 5 minutes, at 3000-4000 rpm, 4°C).
Discard the supernatant. Add 0.1M CaCl2 to pellet and resuspend it. Discard supernatant, add 2ml 0.1M of CaCl2 and resuspend pellet
into it. Prepare the aliquots by adding glycerol and keep in storage at -80°C.
Restriction digestion and Ligation reaction
The restriction digestion of an eluted DNA or PCR purified product (insert) and vector is performed with the same restriction enzymes. Subsequently the ligation reaction is performed using T4 DNA ligase.
Heat Shock Method and Blue White Selection
The delivery of a ligation product into a bacterial cell (a competent E. coli cell) is generally and commonly executed by the heat shock method. Heat shock treatment is given to the competent cell culture after the addition of a ligated product to it at 42°C, for 2 minutes. After heat shock treatment, transformed bacterial cells are incubated in a small quantity of liquid medium without antibiotics, for a short time period. Because of this incubation, the expression of the resistance gene (an enzyme) reaches the level at which this enzyme may detoxify the antibiotic in the culture regime and thereby allows the growth of the transformed bacterial cell on culture plates. The bacterial culture is spread onto an L.B. solid medium of ampicillin along with X-Gal and IPTG. This is done for blue white selection to select a recombinant clone. Cultured plates are incubated at 37°C overnight, and bacterial colonies appear on the culture plate if bacterial transformation is achieved. Keeping the culture plates for a long period of time results in the formation of satellite colonies.
