DNA Cloning: A Hands-on Approach

139 Pages·2019·5.146 MB·English

by  Seok-Yong Choi, Hyunju Ro, Hankuil Yi

Seok-Yong Choi
Hyunju Ro
Hankuil Yi
DNA Cloning:
A Hands-on
Approach
DNA Cloning: A Hands-on Approach
Seok-Yong Choi • Hyunju Ro • Hankuil Yi
DNA Cloning: A Hands-on
Approach
Seok-Yong Choi Hyunju Ro
Department of Biomedical Sciences Department of Biological Sciences
Chonnam National University Medical Chungnam National University
School Daejeon, South Korea
Gwangju, South Korea
Hankuil Yi
Department of Biological Sciences
Chungnam National University
Daejeon, South Korea
ISBN 978-94-024-1660-2 ISBN 978-94-024-1662-6 (eBook)
https://doi.org/10.1007/978-94-024-1662-6
Library of Congress Control Number: 2019932979
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Dedicated to our wives, Eun Young Choi
(Seok-Yong Choi), Christina Do (Hyunju
Ro), and Jeong-Won Nam (Hankuil Yi).
Preface
This book was first published in Korean in 2010 to help undergraduate and graduate
students learn DNA cloning in the lab. This book was not so much about the theo-
retical background of DNA cloning as it was about step-by-step instructions for
DNA cloning. I wrote this book supposing that I was teaching DNA cloning to a
novice sitting at the bench right next to me.
Upon the release of the Korean version of this book, principal investigators (PIs),
graduate students, and undergraduate students, both domestic and overseas, compli-
mented me on the book. I was flattered. Yet as you know, there is no free lunch.
Principal investigators then asked me to publish an English version of this book as
they wanted the foreign graduate students in their labs to learn practical information
about DNA cloning. I laughed it off in the beginning as publishing a book in English
was very daunting to me. As more and more PIs asked me the same thing, I began
to take it seriously. I asked Ms. Aimee Hwang to translate the Korean version into
English, contacted the Springer Publishing company (now Springer Nature
Publishing company) to publish the English version, and revised an English draft
with Professor Hyunju Ro. The revision took far much longer than we expected,
causing us to stop halfway through. After many e-mails from Springer Nature
Publishing reminding us of our deadline, now 3 years overdue, we locked ourselves
(Professors Hyunju Ro and Hankuil Yi and myself) in a hotel room for 4 days and 3
nights to revise the English draft, as a last resort. After a couple of more meetings,
we finally finished the revision.
There is probably much room for improvement in this book. Should you have
any questions or comments, please e-mail me ([email protected]). I will
use them to update the next edition.
I am grateful to Ms. Aimee Hwang for writing the first draft of the English ver-
sion; Professor Hueng-Sik Choi at Chonnam National University for drawing the
cartoons for the book; Professor Hae-Chul Park at Korea University for providing a
BAC recombineering figure; Mr. Gopalakrishnan Chandrasekaran for critical read
of the manuscript; Dr. M. Wayne Davis at the University of Utah for developing the
ApE software, a plasmid editor; and Professor Michael Frohman and Ms. Yelena
Altshuller for opening a door to the cloning field for me.
I have heard that at some universities in North Korea, lectures are given in
English. I hope this book may be used someday for a class in North Korea.
ix
x Preface
DNA cloning is a very creative procedure, and I hope that you’d appreciate it
upon reading this book. Happy cloning!
I will finish the preface by quoting Bert Vogelstein, who sets a milestone in colon
cancer research.
Anyone who liked to play with toys has got to like to do science, because they are the
world’s best toys.
May 1, 2019 On behalf of the authors,
Seok-Yong Choi
Contents
1 What Is Cloning? .................................................................................... 1
1.1 Definition of Cloning ..................................................................... 1
1.2 Discovering a New Gene ................................................................ 1
1.3 Cloning in the Past ......................................................................... 2
1.4 Cloning in the Present .................................................................... 3
2 A Prerequisite for Cloning ..................................................................... 5
2.1 Software Useful for Cloning Design .............................................. 5
2.2 Vector, Plasmid, Construct and the Kozak
Consensus Sequence ...................................................................... 7
2.3 Multiple Cloning Sites (MCS) ....................................................... 10
2.4 Restriction Endonucleases and Star Activity ................................. 11
2.5 Agarose Gel Electrophoresis .......................................................... 17
2.6 Pouring LB Agar Plates .................................................................. 21
2.7 Competent Cells ............................................................................. 22
2.8 The Conversion of Mass of DNA into Molar
Concentration ................................................................................. 25
2.9 Upon Receiving New Plasmids ...................................................... 25
2.10 cDNA Library ................................................................................ 27
References ................................................................................................. 28
3 The First Step in Cloning ....................................................................... 29
3.1 Cut and Paste .................................................................................. 29
3.1.1 RE Selection ....................................................................... 30
3.1.2 Preparation of Vector and Insert for Ligation ..................... 30
3.1.3 Isolation of DNA ................................................................ 31
3.1.4 Dephosphorylation of the Vector ........................................ 32
3.1.5 Ligation .............................................................................. 32
3.1.6 Transformation ................................................................... 33
3.1.7 Verification ......................................................................... 34
3.1.8 DNA Sequencing ................................................................ 36
3.1.9 Making Stock ..................................................................... 37
3.2 DNA Sequencing and Direct Sequencing ...................................... 38
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3.3 PCR and Nested PCR ..................................................................... 40
3.3.1 The Choice of DNA Polymerase ........................................ 41
3.3.2 Designing PCR Primers for Cloning .................................. 41
3.3.3 The Magic Pill of PCR ....................................................... 43
3.3.4 Design of Primers for RT-PCR ........................................... 44
3.3.5 When You Encounter Difficult Cloning,
Use Nested PCR ................................................................. 45
3.4 Fill-In (Full and Partial) ................................................................. 46
3.5 Compatible Cohesive Ends ............................................................ 49
3.6 Methylation .................................................................................... 51
3.7 Three-Piece Ligation ...................................................................... 57
3.8 Site-Directed Mutagenesis ............................................................. 58
3.9 Structure of Plant Transformation Vectors ..................................... 63
3.10 Transformation of Rhizobium radiobacter ..................................... 65
4 The Next Step of Cloning ....................................................................... 67
4.1 How to Insert a DNA Fragment into a Gene .................................. 67
4.2 How to Delete an Internal Region of a Gene ................................. 70
4.3 How to Insert an Epitope Tag into a Gene ..................................... 71
4.4 Translational Fusion vs. Transcriptional Fusion ............................ 75
5 The Last Steps of Cloning ...................................................................... 79
5.1 Method for Cloning Similar Genes in Different Species ............... 79
5.2 RACE (Rapid Amplification of cDNA Ends) ................................ 81
5.3 BAC Recombineering .................................................................... 83
5.4 Old Trick: Partial Digestion ........................................................... 86
5.5 Modification of a Vector ................................................................. 87
5.6 When You Notice a Frame Shift Mutation Upon Cloning ............. 89
5.7 The Reality of Cloning: An Extremely Unlucky Case ................... 91
5.7.1 When Simple Cloning Turns into a Nightmare .................. 91
5.7.2 When Methylation Gave Me a Headache ........................... 96
6 Methods That Make Your Cloning Life Easier .................................... 99
6.1 TA Cloning and Production of a T-Vector ...................................... 99
6.2 TOPO TA Cloning .......................................................................... 103
6.3 Gateway Cloning ............................................................................ 104
6.4 Golden Gate Assembly for Modular Cloning ................................ 106
6.5 In-Fusion Sequence and Ligation-Independent
Cloning (In-Fusion SLIC) .............................................................. 109
6.6 T4 DNA Polymerase Sequence-and Ligation- Independent
Cloning (T4 DNA Pol SLIC) ......................................................... 115
6.7 Non-template PCR Cloning ........................................................... 119
References ................................................................................................. 121