Artificial DNA - Methods and Applications - Y. Khudyakov, H. Fields (CRC, 2003) WW

435 Pages·2003·9.26 MB·English

Artificial
DNA
Methods
and Applications

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CRC PR ESS

Boca Raton London New York Washington, D.C.

Artificial
DNA

Edited by
Yury E. Khudyakov
Howard A. Fields

Methods
and Applications

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International Standard Book Number 0-8493-1426-7
Library of Congress Card Number 2002067416
Printed in the United States of America 1 2 3 4 5 6 7 8 9 0
Printed on acid-free paper

Library of Congress Cataloging-in-Publication Data

Artificial DNA : methods and applications / edited by Yury E. Khudyakov and Howard
A. Fields.
p. cm.
Includes bibliographical references and index.
ISBN 0-8493-1426-7 (alk. paper)
1. DNA—Synthesis. 2. DNA—Derivatives. 3. DNA probes. 4. Genetic engineering. I.
Khudyakov, Yury E. II. Fields, Howard A.

QP624 .A785 2002
660.6′5—dc21
2002067416

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Preface

The only approach to art is art itself.

— Klaus Eidam

The True Life of Johann Sebastian Bach

Research laboratories involved in studies on molecular genetic processes or on the development of
diagnostic, prophylactic, and therapeutic tools for human diseases are all exceedingly familiar with
the use of synthetic oligonucleotides or synthetic DNA. Today, the application of synthetic DNA
is as common as the polymerase chain reaction (PCR), DNA sequencing, or nucleic acid hybrid-
ization. What used to be the exclusive domain for a limited number of research scientists only 20
to 25 years ago has now penetrated into many different applications in research and in health
products in numerous laboratories and biotechnological and pharmaceutical companies. Originally
conceived as a source of artificial genetic material, synthetic DNA has found major applications
in many diverse nongenetic subject areas such as molecular diagnostics, microarray analysis,
computer chip research, and antisense technology, to name only a few. Several factors have
contributed to the current widespread use of synthetic DNA. Some of these factors may be directly
related to the convenience of obtaining synthetic oligo- and polynucleotides, which have become
readily available in large amounts because of the development of revolutionary techniques for DNA
synthesis over the last three decades; however, the major factor that has contributed to the expo-
nential growth and use of synthetic DNA is more related to the unlimited human control over the
structure of synthetic DNA.
This notion should not be a surprise to readers. If we look closely at the history of humankind,
we will understand that gaining control over the environment is the major adaptive mechanism
used by humans. The evolution of humankind may be envisioned as breaking from the grasp of
natural selection to embrace the implementation of rational development directed by humans.
Science is used as a steppingstone to control nature for the benefit of humankind. Being archetypal
to humans, this trend can be readily noted in many other fields of human activity. It is our craving
to exercise control over our lives that has led to the development of an artificial world. For example,
we have constructed artificial means for transportation such as automobiles and an artificial strictly
controlled environment such as highways for the optimal use of automobiles. Consider modern
homes, which are actually entirely artificial entities. They provide the same purpose as caves offered
to our ancestors. However, a dramatic improvement in the quality of living found in modern
buildings can hardly be disputed by anyone who lives in today’s world and who may have had the
opportunity to visit such caves, or who has a vivid imagination to picture such primitive residences.
These simple examples show that the active process of gaining control over our life is, to a large
extent, built on the development of artificial means that mimic nature’s function without directly
mimicking nature’s ways.
Since life itself is predicated on nucleic acids as carriers of genetic information, nucleic acids
as the subject of biological research best exemplify these trends of human activity in the biological
sciences. We began to learn how to manipulate existing genetic material using genetic engineering
techniques. However, only when we learned how to synthesize DNA of any sequence did real
opportunities arise for generating an artificial, strictly controlled DNA world. It has opened new
avenues for designing artificial genetic systems that will help avert harm’s way and promote health
and prosperity for humanity.

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The very first attempt to chemically synthesize short oligonucleotides from nucleotides was
performed in the laboratory of Dr. H. G. Khorana more than 35 years ago. The significance of this
pioneering research in the biological sciences and its enormous potential benefit to the progress of
humankind cannot be overstated or underestimated. These studies provided for the first time access
to unlimited modifications of the genetic material opening a new era for the unrestrained manipu-
lation of the DNA structure, which will ultimately lead to the creation of new artificial genetic
systems with strictly predetermined properties.
History shows that artificial systems may result in producing similar problems as their natural
counterparts. For example, cyberspace has created an opportunity for the generation of parasitic
units of information such as computer viruses and worms that have found very efficient ways to
replicate themselves at the expense of the entire system. We are just now at the doorstep of exploring
the potential use of artificial genetic systems, and, as should have been anticipated, there are already
the first signs that these systems may create their own successfully replicating parasitic genetic
units (see Chapter 4). By creating artificial systems we create a new environment and a new
opportunity for self-generation of parasitic units potentially harmful to this new artificial environ-
ment. This is not the first time, however, that we must balance progress in some areas of human
activity against potential harm this progress may bring. “Understand the harm and benefit in
everything” teaches Miamoto Musashi, a philosopher, artist, writer, and peerless undefeated sword
fighter from 17th century Japan, in his The Book of Five Rings. After putting his life on the line
on numerous occasions in his quest for self-perfection, he came to understand the value of this
notion and tried to pass it on to posterity as a piece of his wisdom.
During more than 30 years of research, synthetic DNA has been developed for use in many
applications, some of which are described in this book. While the harm from using artificial DNA
is still conceptual, we have already witnessed the invaluable fruits this new technology has brought
to reality. Today, the list of applications for synthetic DNA is so lengthy that a cursory inspection
of this list would take more time than the reading of this preface. The myriad uses for synthetic
material are only limited by our own imaginations. As always, the best is still to come.
The history of research studies and applications of synthetic DNA is rather short. However,
even a cursory analysis of the published literature reveals some intriguing trends. A literature search
using the key words “oligonucleotide,” “synthetic gene,” “PCR,” and “microarray” reveals very
limited growth in the number of publications that used the word “oligonucleotide” at the beginning
of 1970s. This growth leveled off throughout the 1970s and 1980s. However, in the late 1980s, the
number of publications started expanding again, but this time growth occurred at an unprecedented
rate until the early 1990s to a level where it was 20 to 30 times greater than in any other previous
year. This dramatic increase was paralleled by an equally steep growth in the number of publications
using the term “PCR.” While the number of publications using the abbreviation “PCR” continued
to increase until the turn of millennium, the number of publications using the word “oligonucleotide”
declined in the mid-1990s, probably as a result of the less frequent use of this word in the context
of PCR primers. This downward trend has been reversed in the last 4 to 5 years coinciding with a
no less impressive growth in the number of articles using the word “microarray.” What this simple
analysis reveals is that the use of oligonucleotides over the last decade has been driven by the
application of oligonucleotides to new technologies such as PCR, sequencing, and microarray.
The use of synthetic genes, however, has followed a very different trend. Unlike many other
technologies, the application of synthetic genes did not rapidly expand after the construction of
the very first one in the mid-1970s. The use of the term “synthetic gene” experienced a very mild
increase in popularity around 1980 to 1981, but this blip on an otherwise flat line may be explained
by the introduction of new efficient chemical methods for oligonucleotide synthesis, which made
research studies on the synthesis and utility of long polynucleotides more affordable. Nonetheless,
the number of publications quickly fell to zero within a couple of years. After the introduction of
automatic synthesizers, which made synthetic oligonucleotides more available, the number of

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publications reporting assembly of synthetic genes started increasing again. Later, the number of
publications using the term “synthetic gene” experienced another mild boost by the application of
PCR technologies, which significantly simplified obtaining long polynucleotides from short syn-
thetic oligonucleotides. However, by the mid-1990s the number of publications began to decline
once again.
Thus, while the use of synthetic oligonucleotides matured into various applications, all examples
demonstrating an increased interest in synthetic genes were associated with oligonucleotide syn-
thesis technology improvement and the development of new approaches to the synthesis of long
DNA molecules, rather than on potential applications. The expansion on the use of synthetic genes
has been hampered for years by the inefficacy of methods to assemble long polynucleotides. Despite
the fact that the application of PCR has significantly improved the efficiency for the synthesis of
long polynucleotides, the use of synthetic genes is still declining in popularity. Therefore, we must
conclude that the use of synthetic genes is not yet application driven. Designing synthetic genetic
systems requires novel integrated approaches based upon a “procreative” rather than “anatomical”
course and a thorough understanding of the study subject. Do we have sufficient knowledge to
rationally design and construct an artificial genetic world? Even if we do not yet possess such
knowledge for many sophisticated applications, are we ready to explore a new world of artificial
genetics? As this book proves, so much already has been done. But, how much will be done in the
future! Breakthroughs may happen at any time. This is a very exciting area of molecular science
that is on the very precipice of a scientific explosion waiting for new explorers. Join in!

Yury E. Khudyakov, Ph.D.
Howard A. Fields, Ph.D.

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The Editors

Yury E. Khudyakov, Ph.D., is Chief, Computational Molecular Biology Activity and Deputy Chief,
Developmental Diagnostic Laboratory, Laboratory Branch, Division of Viral Hepatitis, National
Center for Infectious Diseases (NCID), Centers for Disease Control and Prevention (CDC), Atlanta,
Georgia. He received his M.Sc. in Genetics from the Novosibirsk State University, Novosibirsk,
Russia in 1977 and his Ph.D. in Molecular Biology from the D.I. Ivanovsky Institute of Virology,
Academy of Medical Sciences, Moscow in 1985. He started his research career (1977–1980) in
the Laboratory of Gene Chemistry at the M.M. Schemyakin Institute of Bioorganic Chemistry,
Academy of Sciences, Moscow, one of the first laboratories in Russia to develop new technologies
for chemical synthesis of DNA. He was a Research Fellow (1980–1985) in the laboratory of Viral
Biochemistry and Chief, Genetic Engineering Section (1985–1991) in the laboratory of Chemistry
of Viral Nucleic Acids and Proteins at the D.I. Ivanovsky Institute of Virology, Moscow. In 1991,
he joined the Hepatitis Branch (HB), Division of Viral and Rickettsial Diseases (DVRD)/NCID/
CDC as a National Research Council Research Associate, National Academy of Sciences, U.S.A.
Since 1996 he has served as Chief, Developmental Diagnostic Unit, Molecular and Immunodiag-
nostic Section/HB/DVRD/NCID/CDC.
Dr. Khudyakov’s main research interests are applications of synthetic DNA to the development
of new diagnostics and vaccines, molecular biology and evolution of hepatitis viruses, and bioin-
formatics.
Dr. Khudyakov has published more than 80 research papers and book chapters. He is the author
of several issued and pending patents. He is a member of the editorial board for the Journal of
Clinical Microbiology.

Howard A. Fields, Ph.D., is Chief of the Developmental Diagnostic Laboratory, Hepatitis Laboratory
Branch, Division of Viral Hepatitis, National Center for Infectious Diseases (NCID), Centers for Disease
Control and Prevention (CDC), Atlanta, Georgia. Since 2002, he has been a Distinguished Consultant
for CDC. He received his M.S. in Microbiology from the University of Maine, Orono, in 1971 and
his Ph.D. in Virology from the University of New Hampshire, Durham, in 1974. From 1974 to 1976,
after completing his Ph.D, he did postdoctoral work in immunochemistry at Baylor College of Medicine,
Houston, Texas, after which he joined the Hepatitis Program at the CDC field station located in Phoenix,
Arizona as an immunovirologist. In 1980, Dr. Fields became Chief of the Immunochemistry Laboratory,
Hepatitis and Viral Enteritis Division, CDC. In 1983, the hepatitis program was relocated to Atlanta,
Georgia, where he became Chief of the Molecular and Immunodiagnostic Section within the Division
of Viral and Rickettsial Diseases.
Dr. Fields’ main research interests are biochemical and biophysical characterization of antigens;
epitope mapping of antigens using monoclonal antibodies, Western blots, and synthetic peptides;
development of sensitive and specific immunoassays; development of molecular diagnostic assays;
development of methods to assemble synthetic genes; development of highly efficient procaryotic
and eucaryotic expression systems; development of animal models for viral hepatitis; molecular
epidemiology using nucleotide sequence comparisons and phylogenetic analysis; and development
of artificial genes encoding mosaic recombinant proteins as immunodiagnostic reagents.
Dr. Fields has published 141 research papers and book chapters. He is the author of several
issued and pending patents. He is a past member of the editorial boards for the Journal of Clinical
Microbiology and the Journal of Applied and Environmental Microbiology. At present, Dr. Fields
is an associate editor for the Journal of Clinical Microbiology.

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