Advances and Clinical Practice in Pyrosequencing

398 Pages·2016·20.614 MB·English

by  Guohua Zhou, Qinxin Song (eds.)

Handbooks
Guohua Zhou · Qinxin Song Editors
Advances and
Clinical Practice
in Pyrosequencing
S P H
PRINGER ROTOCOLS ANDBOOKS
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Advances and Clinical Practice
in Pyrosequencing
Editors
Guohua Zhou
Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science,
Medical School of Nanjing University, Nanjing, Jiangsu, China
Qinxin Song
Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education,

China Pharmaceutical University, Nanjing, Jiangsu, China
Editors
Guohua Zhou Qinxin Song
Jinling Hospital Key Laboratory of Drug Quality Control
State Key Laboratory of Analytical and Pharmacovigilance
Chemistry for Life Science Ministry of Education
Medical School of Nanjing University China Pharmaceutical University
Nanjing, Jiangsu, China Nanjing, Jiangsu, China
ISSN 1949-2448 ISSN 1949-2456 (electronic)
Springer Protocols Handbooks
ISBN 978-1-4939-3306-8 ISBN 978-1-4939-3308-2 (eBook)
DOI 10.1007/978-1-4939-3308-2
Library of Congress Control Number: 2015957401
Springer New York Heidelberg Dordrecht London
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Dedication
The book is specially dedicated to Professor Hideki Kambara, an unsung hero of the human
genome project.

Prefa ce
The high-throughput DNA sequencing method based on Sanger’s principle provided an
effi cient tool for the sequencing of the whole human genome. With the help of capillary
array platform invented by Professor Kambara, the human genome project was fi nished two
years earlier than expected. In the post-genomic era, many biomarkers related to early dis-
ease diagnosis and personalized medicine have been found by the use of next-generation
DNA sequencers. Although there are several DNA sequencing tools available for clinical
detection of these biomarkers, pyrosequencing has an especially important role in identify-
ing microbial genotypes, as well as in sequencing a short fragment containing the biomark-
ers related to personalized medicine.
Pyrosequencing is a sequencing-by-synthesis method based on the bioluminometric
detection of by-products of inorganic pyrophosphates (PPi) during primer extension reac-
tion. Although only several tens of bases can be sequenced during a run, it is enough for
clinical routine use in detecting known biomarkers. As pyrosequencing is very suitable to
the sequencing of short DNA sequence, the CFDA approved pyrosequencing technology
for clinical use. Pyrosequencing is becoming the most popular DNA sequencing tool for
personalized medicine in hospitals.
I have engaged in pyrosequencing research since 1999 when I came to Professor
Kambara’s research group in Central Research Laboratory of Hitachi. Professor Kambara
directed me to perform pyrosequencing research in developing new chemistry for increas-
ing the sensitivity, simplifying protocols for SNP typing, and miniaturizing the instrumen-
tation. Professor Kambara, who is honored as an unsung hero of the human genome
project by Science, is a highly reputed scientist in the DNA sequencing research fi eld. I am
very fortunate to have had his supervision in the research of DNA detection. Even after
my return to China from Japan, he supported me in following pyrosequencing research
for many years.
The aim of this book is to improve pyrosequencing protocols as well as instrumenta-
tion for better clinical use. Due to our continuous contributions, pyrosequencing is greatly
improved in terms of template preparation, sensitivity, instrumentation, and detectable
target species. We simplifi ed the protocols of pyrosequencing by skipping the ssDNA prep-
aration step. We enabled pyrosequencing to quantify the expression levels of mRNA and
microRNA by coupling base barcodes into pyrosequencing. We adapted pyrosequencing
to prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes of fetal
genomic DNA. We miniaturized the pyrosequencer by using a photodiode array as the
light sensor. This book describes all of these improvements and novel applications of pyro-
sequencing technology.
There are 34 chapters in the book, and they are grouped as fi ve parts: Part I is
Advances in Pyrosequencing Template Preparation; Part II explores Pyrosequencing
Technology Innovations; Part III delves into Multiplex Pyrosequencing based on bar-
codes; Part IV looks at Miniaturizing Pyrosequencing Equipment; and Part V examines
Applications.
vii
viii Preface
Now we are progressing toward precision medicine. To allow the right drug with the
right dose at the right time, genotyping of an individual patient is required before prescrib-
ing a drug. I believe that pyrosequencing would be a useful tool in this progress. This book
should be useful for people who are engaged in personalized medicine, disease control, and
DNA diagnosis in other fi elds.
Professor Kambara, my teacher, retired from Hitachi in March 2015. I would like to
dedicate this book to him, a great contributor to DNA sequencing technology.
Nanjing, China G uohua Z hou
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
PART I TECHNOLOGY PREPARATION
1 Preparation of Single-Stranded DNA for Pyrosequencing by LATE-PCR. . . . . 3
Huiyong Yang, Chao Liang, Zhiyao Chen, Bingjie Zou, Qinxin Song,
and Guohua Zhou
2 A Simplified Protocol for Preparing Pyrosequencing Templates
Based on LATE-PCR Using Whole Blood as Starting Material Directly. . . . . . 13
Yunlong L iu , H aiping Wu , H ui Y e , Z hiyao C hen , Qinxin Song ,
Bingjie Z ou , J ianzhong R ui , and Guohua Z hou
3 Improvement of LATE-PCR to Prepare Pyrosequencing Template . . . . . . . . . 2 3
Qinxin S ong , Huiyong Y ang , B ingjie Z ou , Hideki K ambara ,
and G uohua Z hou
4 Pyrosequencing Templates Generated by Nicking PCR Products
with Nicking Endonuclease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Qinxin S ong , Haiping Wu , Guohua Z hou , and Hideki K ambara
5 Pyrosequencing Templates Generated by Asymmetric Nucleic Acid
Sequence-Based Amplification (Asymmetric-NASBA) . . . . . . . . . . . . . . . . . . . 4 1
Huning J ia , Zhiyao C hen , H aiping Wu , H ui Ye , Bingjie Z ou ,
Qinxin S ong , and G uohua Zhou
6 Gel Immobilization of Acrylamide-Modified Single-S tranded
DNA Template for Pyrosequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Pengfeng X iao , H uan Huang , B ingjie Zou , Q inxin Song ,
Guohua Z hou , and Zuhong L u
7 Multiplex PCR Based on a Universal Biotinylated Primer
to Generate Templates for Pyrosequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Zhiyao C hen , Y unlong L iu , Hui Y e , H aiping W u , Jinheng L i ,
Bingjie Z ou , Q inxin S ong , and Guohua Zhou
PART II TEMPLATE INNOVATION
8 A Novel Pyrosequencing Principle Based on AMP–PPDK Reaction
for Improving the Detection Limit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 9
Guohua Z hou , T omoharu Kajiyama , Mari Gotou , Akihiko Kishimoto ,
Shigeya S uzuki , and Hideki K ambara
9 Pyrosequencing Chemistry Coupled with Modified Primer Extension
Reactions for Quantitative Detection of Allele Frequency
in a Pooled Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5
Guohua Z hou , M asao Kamahori , K azunori Okano , G ao Chuan ,
Kunio H arada , and Hideki Kambara
ix