Recent Patents on DNA & Gene Sequences 2012, 6, 10-21

10

Recent Patents on Oligonucleotide Synthesis and Gene Synthesis

Tingsheng Yua,*, Xiao Baoa, Wenxian Piaoa, Jingli Penga, Wei Lia, Cui Yanga, Meng Xinga,
Yiliu Zhanga, Jinhuan Qia, Lei Xua, Li Xu and Qiuyun Liu*
The Key Laboratory of Gene Engineering of Ministry of Education and Biotechnology Research Center, The School of
Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
Received: August 17, 2011

Revised: October 03, 2011 Accepted: October 24, 2011

Abstract: Gene synthesis is an emerging field which has widespread implications in synthetic biology and molecular biology. The field is constantly evolving which has led to key advances in oligonucleotide synthesis and gene synthesis
technologies, with simplicity, cost effectiveness and high throughput. The miniaturization, multiplexing, microfluidic
processing and the integrated microchip engineering will drive down cost and increase productivity without compromising DNA synthesis fidelity, whereas the gigantic amount of genome information provides infinite source of DNA elements and genes as raw material for synthetic biology. This article describes some of the recent patents on oligonucleotide
synthesis and gene synthesis.

Keywords: Oligonucleotide synthesis, gene synthesis, miniaturization, multiplexing, microfluidic processing, microchip.
INTRODUCTION
Oligonucleotide synthesis and DNA synthesis are integral parts of the gene synthesis processes. Oligonucleotide
synthesis is the process of attaching short nucleotides into a
string, using chemical methods. It is essential in producing
primers for polymerase chain reaction (PCR) [1]. Typically,
oligonucleotides are synthesized mainly by means of phosphodiester synthesis, phosphotriester synthesis and so on.
However, the phosphoramidite method emerged to be the
most desirable and most popular approach to synthesize oligonucleotides [2].
In the post-genomic era, the large-scale scrutiny of genome and proteome, and the deliberation, comparison and
integration of mega data sets, became routine in biological
laboratories. DNA sequences from unknown proteins are
essential in exploring and building inventories on the functions of these proteins. The canonical way of gene cloning
from various sources is pretty straightforward, but the pertinent approaches have several limits: the expression of natural gene in heterologous systems like Escherichia coli or else
may not be ideal; vaccine attenuation may require codon deoptimization, and druggable proteins may need to be engineered for longer half-lives and more potent activities, etc.
Moreover, directed evolution may require de novo gene synthesis, and creation of artificial life forms requires mega
DNA synthesis. With rapid advances in synthetic biology
and molecular biology, the demand for synthetic nucleic
acids is on the rise. Besides, the techniques of microarrays
*Address correspondence to these authors at the School of Life Sciences,
Sun Yat-sen University, Guangzhou, 510275, P. R. China; Tel: 86-2084110296; E-mail: yts9009@sina.com; and The Key Laboratory of Gene
Engineering of Ministry of Education, Sun Yat-sen University, Guangzhou
510275, P. R. China; Tel: 86-20-84110296; Fax: 86-20-84036551;
E-mail: liuqiuyunzhongda@yahoo.com.cn;
a

and polymerase chain reaction (PCR) also accelerated the

widespread adoption of synthetic nucleic acid polymers [3].
Below we will review some of the recent patents on oligonucleotide synthesis and gene synthesis.
PART I OLIGONUCLEOTIDE SYNTHESIS
Phosphoramidite method, which was based on the work
of Caruthers, is routinely used as a standard approach for
preparing synthetic nucleic acids. H-phosphonate [4], on the
other hand, even though achieving the same goal of synthesizing the desired polymer, fails to attract due attention compared to the former one due to lower yield.
To streamline the procedure and automate the processes,
solid phases are commonly employed to allow the growing
molecular chain to anchor on. Since the polymer has to be
split off upon completion, suitable linkers, which can connect the polymer and its solid phase, are also indispensable
[5]. In the 1990s, with the advent of in situ synthesis of microarrays, which could load thousands of different sequences
on a substrate on a single try, many more methods have
flourished from it and synthetic nucleic acid research advanced to another level [5] (Table 1).
Chloral-Free Dichloroacetic Acid in Oligonucleotide Synthesis [6]
Oligonucleotides and derivatives have at least a number
of applications in research and medicine. Oligomers can
serve as primers for PCR amplifications and probes for DNA
and RNA hybridizations, as well as for next generation sequencing by hybridizations. Oligonucleotide chips are
widely used in the studies of transcriptome, epigenome, as
well as genome hybridizations such as genome tiling. Oligonucleotides are also invaluable as therapeutics in the
clinic.

These authors contributed equally to this work

2212-3431/12 $100.00+.00

2012 Bentham Science Publishers

Recent Patents on Oligonucleotide Synthesis and Gene Synthesis

Table 1.

Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

11

Patents Related to Oligonucleotide Synthesis

Publication Number

Ref. No. Title

Inventors

Publication
Date

US8008270

Antiviral oligonucleotides targeting viral families

Vaillant, A.; Juteau, J.M.

2011/08/30

US8008269

Antiviral oligonucleotides

Vaillant, A.; Juteau, J.M.

2011/08/30

US8008468

RNAi expression constructs with liver-specific enhancer/promoter

Roelvink, P. W. ; Suhy, D. A.; (+3)

2011/08/30

US8008005

Method for the synthesis of DNA sequences

Belshaw, P.J; Sussman, M.J.(+2)

2011/08/30

US20110196145A1

Process for desilylation of oligonucleotides

Manoharan, M.;
Jung, M. E.(+3)

2011/08/11

US7985565

Method of nucleic acid amplification

Kawashima, E.H.; Farinelli, L.; (+1)

2011/07/26

US20110177514A1

Integrated instrument performing synthesis and amplification, and a system and method thereof

Froehlich, T.; Gutekunst, M.(+3)

2011/07/21

US7981871

Modified macromolescules and associated methods of synthesis and use

Prestwich, G.D.;
Shu, X.Z.; (+1)

2011/07/19

US20110171649A1

Detection of nucleic acids by oligonucleotide probes cleaved in presence

of endonuclease V

Kutyavin, I.;

2011/07/14

US7972820

Isothermal amplification of nucleic acids on a solid support

Mayer, P.

2011/07/05

US7964343

Method for rapid purification of nucleic acids for subsequent analysis by

mass spectrometry by solution capture

Hofstadler, S.A.; Cummins, L.L.

2011/06/21

US20110137021A1

Sulfur transfer reagents for oligonucleotide synthesis

Guzaev, A.P.;

2011/06/09

US7947659

iRNA agents targeting VEGF

Fougerolles, A.D.;
Kamenetsky, M.F.; (+3)

2011/05/24

US20110124524A1

Fluid processing device for oligonucleotide synthesis and analysis

Ermakov, S. V.

2011/05/26

US7939258

Nucleic acid amplification procedure using RNA and DNA composite

primers

Kurn, N.; Wang, S.

2011/05/10

US7939677

Oligomeric compounds comprising 4'-thionucleosides for use in gene

modulation

Bhat, B.; Dandc, P.; (+4)

2011/05/10

US7939256

Composition and method for nucleic acid sequencing

Williams, J.G.K.

2011/05/10

US20110097762A1

Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports

Gao, X.; Zhang, H.; (+5)

2011/04/28

WO2010134992A3

Synthesis labile base protected modified deoxy & modified ribonucleosides, corresponding phosphoramidites and supports and their use in high
purity oligonucleotide synthesis

Srivastava, S.C; Srivastava, N.P

2011/04/07

US7919612

2'-substituted oligomeric compounds and compositions for use in gene

modulations

Baker, B.F ; Eldrup, A.B. ; (+6)

2011/04/05

US7919473

IRNA agents targeting VEGF

Fougerolles, A.D.; Kamenetsky,

M.F.; (+3)

2011/04/05

WO2011028218A1

Process for triphosphate oligonucleotide synthesis

Zlatev, I.; Morvan, F.; (+3)

2011/03/10

US7901892

Methods and compositions for determining the purity of chemically synthesized nucleic acids

Agris, P. F. ; Mitchell, L. G.; (+1)

2011/03/08

US20110046344A1

Parallel preparation of high fidelity probes in an array format

Kuimelis, R. G. ;
Mcgall, G. H.

2011/02/24

US20110045541A1

Method of nucleic acid amplification

Rudi, K.; Holck, A.

2011/02/24

US7893249

Deprotection and purification of oligonucleotides and their derivatives

Bowman, K; Shaffer, C.; (+1)

2011/02/22

12 Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

Yu et al.

(Table 1) contd.

Publication Number

Ref. No. Title

Inventors

Publication
Date

US7893036

In vivo production of small interfering RNAs that mediate gene silencing

Zamore, P.D.; Juanita, M.; (+3)

2011/02/22

US7884086

Conjugates for use in hepatocyte free uptake assays

Bennett, F. C.;
Mckay, R. (+6)

2011/02/08

US7875733

Oligomeric compounds comprising 4'-thionucleosides for use in gene

modulation

Bhat, B.; Dandc, P.; (+4)

2011/01/25

US20110015382A1

Synthesis of N-FMOC protected deoxy nucleosides, ribo nucleosides,

modified deoxy and ribo nucleosides, and phosphoramidites, and their use
in oligonucleotide synthesis

Srivastava, S.C.; Srivastava, N.P.

2011/01/20

US7872121

Process for the removal of exocyclic base protecting groups

Nanda, D.S;
Satya, K

2011/01/18

US20110009606A1

Synthesis of 2', 3'- and 3', 5' cyclic phosphate mono-and oligonucleotides

Laikhter, A;
Srivastava, S.C; (+1)

2011/01/13

US20110009294A1

Methods for genotyping selected polymorphism

Jones, K.W. ;
Shapero, M. H.; (+1)

2011/01/13

US7862820

Immunoglobulin chimeric monomer-dimer hybrids

Peters, R.T.;
Mezo, A.R.; (+3)

2011/01/04

US7858560

Capture compounds, collections thereof and methods for analyzing the

proteome and complex compositions

Koster, H.;
Siddiqi, S.; (+1)

2010/12/28

US7858311

Composition and method for nucleic acid sequencing

Williams, J. G. K.

2010/12/28

US7851615

Lipophilic conjugated iRNA agents

Manoharan, M.;
Rajeev, K. G.

2010/12/14

US7846733

Methods and compositions for transcription-based nucleic acid amplification

Kurn, N.;
Palo, A.C.

2010/12/07

US7846666

Methods of RNA amplification in the presence of DNA

Kurn, N.;
Palo, A.C.

2010/12/07

WO2010134992A2

Synthesis labile base protected modified deoxy & modified ribonucleosides, corresponding phosphoramidites and supports and their use in high
purity oligonucleotide synthesis

Srivastava, S.C.; Srivastava, N.P.

2010/11/25

US7838466

Device for chemical and biochemical reactions using photo-generated

reagents

Gao, X.;
Zhou, X.(+1)

2010/11/23

US7834171

Modified polynucleotides for reducing off-target effects in RNA interference

Leake, D.;
Reynolds, A.R.; (+2)

2010/11/16

EP2248820A1

Universal supports for oligonucleotide synthesis

Azhayev, A.; Antopolskii, M.

2010/11/10

WO2010062404A3

N-fmoc nucleosides and phosphoramidites, and oligonucleotide synthesis

Srivastava, S.C.; Srivastava, N.P.

2010/10/14

US7812149

2'-Fluoro substituted oligomeric compounds and compositions for use in

gene modulations

Prakash, T. P.;
Baker, B. F. (+6)

2010/10/12

US7807807

Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports

Gao, X.;
Zhang, H.; (+5)

2010/10/05

US7803529

Solid phase sequencing of biopolymers

Cantor, C. R.;
Koster, H.; (+2)

2010/09/28

US7794945

Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

Hedgpeth, J.;
Afonina, I. A.; (+4)

2010/09/14

Recent Patents on Oligonucleotide Synthesis and Gene Synthesis

Antisense oligonucleotides are capable of silencing gene

expression, thus reducing protein levels. It has held great
promise in pre-clinical research. Its mode of action is distinct
from conventional therapeutic methods, which usually
modulate protein activities through direct interaction between putative drugs and drug targets-usually proteins in
nature.
A typical oligonucleotide synthesis using phosphoramidite chemistry (i.e. the amidite methodology) includes a solid
primer support. The 5-hydroxyl protecting groups in the
nucleoside linked to the solid support need to be removed in
the synthesis of oligonucleotides. The 4, 4-dimethoxytriphenylmethyl (DMT) group has been frequently used at
this position, and it can be cleaved with the use of
dichloroacetic acid (DCA). However, DCA frequently
contains trace amount of chloral or chloral hydrate, and even
1 % residue can generate a lot of chloral adducts on the
oligos. DCA essentially free of impurities was prepared via
vacuum distillation by patentees. The procedure can be
adopted for producing oligonucleotides that are virtually free
of chloral adducts (Table 2).
Activators for Oligonucleotide and Phosphoramidite
Synthesis [7]
The use of aryl-substituted 5-phenyl-1H-tetrazoles, with
perfluoroalkyl groups on the aromatic ring as activators in
the synthesis of oligonucleotides and nucleoside phosphoramidites, was documented in this patent. The activators were
highly soluble and efficient, and the coupling time for DNA
phosphoramidites was about or less than 15 seconds. The
coupling time for 2-O-tert-butyldimethylsilyl RNA phosphoramidites was no more than 5 minutes. A nucleoside or
oligonucleotide harbouring a hydroxyl group, could also
react with a phosphitylating agent in the presence of the activator mentioned above to generate a phosphoramidite.

Fig. (1). The general structure of novel aryl-substituted 5-phenyl1H-tetrazoles as catalysts in the coupling reactions of the
phosphoramidite approach. At least one R contains a perfluoroalkyl
substituent and n is an integer selected from 1-5. Adapted from
reference [7].

A spectrophotometric DMT assay showed that coupling

efficiencies over 99.0% could be achieved in the synthesis of
oligonucleotide. The purity of the crude oligonucleotides
could peak to 98% under mild conditions.
Enhanced Antisense Oligonucleotides [8]
The gap-widened antisense oligonucleotides, each 18 to
24 nucleotides in length, have a wing-gap-wing linear struc-

Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

13

ture, and improved therapeutic efficacy as compared to 5-105 MOE (2-O-methoxyethyl, 2-MOE or simply MOE) gapmer antisense oligonucleotides targeted at the same sequence. The gap region has 12 to 18 straight 2-deoxyribonucleosides, whereas the two wings have 1 to 4 2-O-(2methoxyethyl) ribonucleotides.

Fig. (2). Formula for a universal support. Adapted from reference

[10].

Sulfur Transfer Reagents for Oligonucleotide Synthesis [9]

Oligonucleotide phosphorothioates are oligonucleotide
analogues whose non-bridging oxygen atoms of the
phosphate group are substituted by a sulfur atom. The
enhanced nuclease resistance has increased their values in
pharmaceutical research. Sulfurization on the phosphite
moiety is allowed after each coupling in the phosphoramidite
approach. Patentee introduced sulfur transfer reagents capable of converting P(


) internucleosidic linkages to P()
phosphorothioate linkages in oligonucleotides.
A widely used Beaucage reagent 1, 2-benzodithiol-3-one1, 1-dioxide, and a compound 5-ethoxy-3H-1, 2, 4dithiazole-2-one abbreviated as EDIT, both with poor hydrolytic stability, were somewhat tricky to synthesize. Agents
such as TETD (tetraethylthiuram disulfide) have displayed
sluggish reaction kinetics hence are less convenient in industrial mass production.
N-formamidino-5-amino-3H-1, 2, 4-dithiazole-3-thiones
were described as efficient sulfur-transfer reagents in this
patent, having clear edge over commercially available Beaucage reagent and TETD. The novel sulfurizing agents were
synthesized inexpensively via simple chemical methods, and
were highly stable in solution. The sulfur transfer from these
reagents was investigated in the solid-phase synthesis of oligonucleotide phosphorothioates via phosphoramidite methods. The efficiency of the sulfur transfer reaction for 2'deoxyoligonucleotides was determined to top 99.5%.
Universal Supports for Oligonucleotide Synthesis [10]
Most oligonucleotide syntheses are conducted on supports pre-attached with a nucleoside. Therefore four deoxynucleoside and four ribonucleoside supports are required
for routine DNA and RNA synthesis.
Currently the use of universal supports for DNA and
RNA synthesis is not popular in industry. One major hurdle
is to find proper conditions to remove the terminal phosphate
attached to the terminal hydroxyl group in the first phosphoramidite reaction cycle.

14 Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

Table 2.
Reference No.

[5]

Yu et al.

Applications and Drawbacks of the Described Patents

Title

Multiplexed sites for

polymer synthesis

[6]

Chloral-free DCA in
oligonucleotide synthesis

[7]

Activators for oligonucleotide and

phosphoramidite
synthesis

Publication
Date

2010-10-14

2010-07-20

2011-03-01

Publication
Number

Inventors

Applications

Drawbacks

WO2010118264

Rinderknecht, D.;
Keen, R.;
Charib, M.

Multiplexing increased productivity for polymer synthesis

Light irradiation for

polymerization or
detachment from reaction sites need to be
highly efficient.

Krotz, A.; Capaldi, D.C.;Gaus,

H.J.J.; Turney, B.

It produced oligonucleotides
that were virtually free of
chloral adducts consequently
led to better purity. Hence,
such oligos would be useful
in therapeutic approaches.

It requires an extra step

of vacuum distillation
for production of chloral-free dichloroacetic
acid.

US7897758B2

Wolter, A.;
Leuck, M.

The activators as catalysts in

coupling reactions of the
phosphoramidite approach
were highly efficient giving
rise to high purity of oligonucleotides.
Gap-widened antisense oligonucleotides have enhanced
activity over conventional
gapmer antisense oligonucleotides

Bioavailability may be
slightly decreased due
to large molecular
weight.

US7759480

[8]

Enhanced antisense
oligonucleotides

2011-04-05

US7919472B2

Monia, B.P.;
Siwkowski, M.;
Bhanot, S.

[9]

Sulfur transfer reagents for oligonucleotide synthesis

2010-05-25

US7723528

Guzaev, A.P.;

It produced stable nucleotide

analogues, Oligonucleotide
phosphorothioates.

Special sulfur transfer

reagents have to be
prepared

US20050250116A1

Azhayev, A.;
Antopolskii, M.

One solid support for all

nucleotides is an advantage.
The supports described permitted fast cleavage and
dephosphorylation under mild
conditions

Efficiencies may vary

for different nucleotides

WO2010094772

Stahler, P. F.;
Carapito, R.;
Stahler, C. F.;
(+4)

Error free genes could be

generated.

Next generation sequencer was needed.

US2005/0272042A1

Gardner, S.N.;
MariellaMariella,
R.P.; Christian,
A.T.; Young,
J.A.; Clague,
D.S.

The use of tetramers was

economical as 256 tetramers
can form any sequence.

Robotics may be
needed for long sequence assemblies.
Assembly using short
oligomers may be very
inefficient at difficult
regions.

US2005/0255477A1

Carr, P.A.;
Chow, B.Y.;
Jacobson, J.M.;
Mosley, D.W.;
Emig, C.

High fidelity sequences may

be obtained via multiple
approaches

Sophisticated equipment may add up gene

synthesis cost.

US2008/0261833A1

Stemmer,
W.P.C.;
Crameri, A.

The use of oligonucleotides

provided unlimited diversity
to the parental sequences for
PCR shuffling and assembly

Efficient screening
method may be an
advantage to deal with
generated libraries.

[10]

Universal supports for

oligonucleotide synthesis

[18]

Synthesis of Sequence-verified nucleic acids

[20]

Sequential addition of
short DNA oligos in
DNA-polymerasebased synthesis reactions

[21]

Methods for high

fidelity production of
long nucleic acid
molecules

[22]

Methods for generating polynucleotides

having desired characteristics by interactive selection and
recombination

2005-11-10

2009-02-20

2005-12-08

2005-11-17

2008-10-23

Recent Patents on Oligonucleotide Synthesis and Gene Synthesis

Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

15

(Table 2) contd.
Reference No.

Title

[23]

Synthetic nucleic acid

molecule compositions and methods of
preparation

[24]

Method for the complete chemical synthesis and assembly of

genes and genomes

[25]

Integrated microfluidic device for gene

synthesis

[26]

Gene synthesis
method

Publication
Date

Publication
Number

Inventors

Applications

Drawbacks

US7906282B2

Wood, K.V.;
Gruber, M.G.;
Zhuang, Y.;
Paguio, A.

The removal of transcriptional regulatory sites enhanced gene expression.

Protein highly expressed may be

slightly different in
primary sequence from
its natural form.

EP1538206

Evans, G,A.

Integrating gene parts and

functional elements, via the
use of simple computer software, was Capable of generating any sequence

Ligation only approach

may not be able to
generate long sequences without PCR

2011-05-26

US20110124049

Li, M.; Ying,

J.Y.; Huang, M.;
Pin Ng, S.

It presented an integrated
microfluidic device for gene
assembly, purification and
error correction.

The oligo design required Tm optimization.

2009-05-11

WO2010132019

Li, M.H.; Ying,

Y.J.; Wai, C.C.;
Bode, M.

The one-step gene synthesis

method was efficient yet
inherently simple.

The oligo design required Tm optimization.

2011-03-15

2005-06-08

Previous universal supports that use neighboring aminomethyl or diamino-ethyl groups to assist the elimination
reaction have been reported by Andrei [9]. All common types
of oligonucleotides can be prepared with these supports. Oligomers with base labile nucleoside units could be synthesized as well. Yet, basic volatile reagents at high temperatures and long treatment period were required in the process.
Such features render these supports somewhat unpopular for
industrial purposes.
In brief, an ideal universal support would allow efficient
cleavage and dephosphorylation at low temperatures and
under relatively mild reaction conditions. The supports described by Alex et al. do not have a pre-attached nucleoside,
and therefore are compatible with any DNA or RNA synthesis. Furthermore, the supports described permit fast cleavage
and dephosphorylation under mild conditions (e.g., using a
2M ammonia in methanol reagent).
In the formula described in Fig. (2), hydrogen, alkyl,
aryl, or a polymeric or silica base material could be selected
for Substituent A, whereas acyl, aroyl, or a polymeric or
silica base material constituted Substituent B. Substituent C
was chosen from a dimethoxytrityl group or a protecting
group removable under acidic or neutral conditions. The
polymeric or silica base material comprised one of Substituent A or B. An oligonucleotide was linked to the support at
substituent C.
PART II GENE SYNTHESIS
A Brief History of Gene Synthesis
Khorana and Itakura pioneered the field with the thenepic total synthesis of tRNA structural genes and the synthesis and expression of the somatostatin gene [11-13]. Polymerase cycling assembly (PCA) has been most popular among
all methods due to its inherent simplicity. Overlapping and
complementary oligonucleotides were denatured, annealed

and recursively extended with a thermal-stable DNA polymerase to give rise to a full-length sequence, which was then
amplified by conventional PCR. PCA, first reported for synthesis of the 303-bp HIV-2 Rev gene, has since evolved into
a widely recognized general method for synthesis of genes of
various sizes [14].
To produce a completely new molecule is both time consuming and expensive. Thus, there is room for a technology
that allows the creation of novel DNA molecules in a single
step without resorting to any existing recombinant or naturally-occurring DNA.
Stemmer et al. split the entire sequence of a gene, designed oligonucleotides of 40 base long that overlapped each
other by 20 bases. The full-length gene was generated in a
single reaction by iterative overlap extension PCR (OEPCR), followed by second PCR amplification with two outer
most primers [15]. The method of Stemmer et al. has since
been recognized for its simplicity and low cost.
Researchers have also been committed in synthesizing
relatively long genes. Lo-Chun Au et al. assembled short
oligonucleotides via ligase chain reaction (LCR) in high
stringency conditions to make unit fragments which were
then joined to give rise to a full-length gene sequence by
PCR, and a recombinant leptin gene was synthesized according to the codon bias of E. coli, so that the procedure did not
confer constraints on the nature of the sequence and length
[16]. Several softwares have been adopted in assisting oligo
design. Marcus Bode, Samuel Khor, Hongye Ye et al. introduced a computer program entitled TmPrime to design oligonucleotide sets for gene assembly by both ligase chain
reaction (LCR) and polymerase chain reaction (PCR) [17].
The program split the long target DNA sequence, and optimized the length of oligonucleotides to achieve homologous
melting temperatures. It presented no constraints on the gene
and oligonucleotide lengths. The list of gene synthesis patents is in Table 3.

16 Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

Table 3.

Yu et al.

Patents on Gene Synthesis

Publication Number

Title

Inventors

Publication
Date

US7932025

Methods for high fidelity production of long nucleic acid molecules with
error control

Carr, P.A.; Chow, B.Y.; (+3)

2011-04-26

US7879580

Methods for high fidelity production of long nucleic acid molecules

Carr, P.A.; Chow, B.Y.; (+3)

2011-02-01

US8008005

Method for the synthesis of DNA sequences

Belshaw, P. J. Sussman,
M.J.(+2)

2011-08-30

US7853410

Method for making polynucleotides having desired characteristics

Selifonov, S.A.; Stemmer,

W.P.C.; (+6)

2010-12-14

US7795030

Methods and compositions for cellular and metabolic engineering

Minshull, J.; Stemmer, W.P.C.

2010-09-14

US7981614

Methods for generating polynucleotides having desired characteristics by

iterative selection and recombination

Stemmer, W.P.C.; Crameri, C.

2011-07-19

US7904249

Methods for identifying sets of oligonucleotides for use in an in vitro

recombination procedures

Selifonov, S.A.; Stemmer

W.P.C.; (+6)

2011-03-08

US7868138

Methods for generating polynucleotides having desired characteristics by

interative selection and recombination

Stemmer, W.P.C.; Crameri, C.

2011-01-11

US7803934

Parallel preparation of high fidelity probes in an aray format

Mcgall, G.H.; Kuimelis R.G.

2010-09-28

US7790381

Method for creating polynuceotide and polypeptide sequences

Arnold, F.; Shao, Z.; (+1)

2010-09-07

US20110190163A1

Genome-Wide Construction of Schizosaccharomyces Pombe Heterozygous Deletion Mutants Containing Gene-Specific Barcodes by the Methods of 4-round Serial or Block PCR, or Total Gene Synthesis Thereof

Hoe K.L.; Kim D.U.; (+15)

2011-08-04

US7957912

Methods for Identifying and producing polypeptides

Selifonov, S.A.; Stemmer,

W.P.C; (+6)

2011-06-07

US7873499

Methods of populating data strucures for use in evolutionary simulations

Selifonov, S.A.; Stemmer,

W.P.C

2011-01-18

US7851679

Reproductive ablation constructs

Rottmann, W.H.; Caneda, K.N.;

(+1)

2010-12-14

US7816085

Method for in vitro melecular evolution of protein function

Carlsson, R.; Hager, C.M.; (+2)

2010-10-19

WO2011102796A1

Novel synthetic zinc finger proteins and their spatial design

Nurmemmedov, E.

2011-08-25

WO2011066185A1

Microfluidic devices and method for gene synthesis

Chu, L.L.

2011-06-03

WO2010130824A3

Collections and uses thereof

Enzelberger, M.; Prassler, J.;

(+1)

2011-01-27

US20110124049A1

Integrated microfluidic device for gene synthesis

Li, M.H.; Ying, J.Y.; (+2)

2011-05-26

US7993921

Cell cycle regulation and differentiation

Dugas, J.; Barres, B.A.

2011-08-09

US7985840

Synthetic antibody phage libraries

Fuh, G; Sidhu, S.S

2011-07-26

US7985575

Single protein production in living cells facilitated by a messenger RNA

interferase

Inouye, M.; Zhang, J.; (+1)

2011-07-26

US7959926

Methods for expression and purification of recombinant human growth

hormone mutants

Buechler, Y; Lieu, R.; (+4)

2011-06-14

US7928215

Methods of screening for compounds that inhibit the biosynthesis of GPI

in malaria parasites

Hata, K; Ogawa, K; (+6)

2011-04-19

US7919591

Modified human plasma polypeptide or Fc scaffolds and their uses

Sheffer, J.; Norman, T.; (+6)

2011-04-05

US7897359

Cell cycle regulation and differentiation

Kocken, C.H.M.K; Thmas,

A.W.; (+3)

2011-03-01

Recent Patents on Oligonucleotide Synthesis and Gene Synthesis

Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

17

(Table 3) contd.

Publication Number

Title

Inventors

Publication
Date

US7883866

Compositions of aminoacyl-tRNA synthetase and uses thereof

Paulsel, A.; Ho, S.C.

2011-02-08

EP2285974A2

Gene synthesis by convergent assmbly of oligonuclotide subsets

Coope, R.; Horspool D.; (+1)

2011-02-23

EP2268808A1

Genome-Wide Construction of Schizosaccharomyces Pombe Heterozygous Deletion Mutants Containing Gene-Specific Barcodes by the Methods of 4-round Serial or Block PCR, or Total Gene Synthesis Thereof

Hidekihama, T.; Hiromichi, Y

2011-01-05

WO2010132019A1

Gene synthesis method

Li, M.H; Ying, Y.J; (+2)

2010-11-18

US7858344

Compositions of aminoacyl-tRNA synthetase and uses thereof

Paulsel, A.; Ho, S.C.

2010-12-28

US7846689

Compositions of aminoacyl-tRNA synthetase and uses thereof

Paulsel, A.; Ho, S.C.

2010-12-07

US 2010/0035768 A1

Methods for in vitro joining and combinatorial assembly of nucleic acid

molecules

Hamilton, O.S; (+3)

WO 2009/103027 A2

Methods for in vitro joining and combinatorial assembly of nucleic acid

molecules

Daniel G. G.; (+3)

US7838265

Compositions of aminoacyl-tRNA synthetase and uses thereof

Paulsel, A.; Ho, S.C.

2010-11-23

US7838219

Method of increasing complementarity in a heteroduplex

Padgett, H.S.; Lindbo, J.A.; (+2)

2010-11-23

US7833759

Method of increasing complementarity in a heteroduplex

Padgett, H.S.; Lindbo, J.A.; (+1)

2010-11-16

US7829310

Compositions of aminoacyl-tRNA synthetase and uses thereof

Paulsel, A.; Ho, S.C.

2010-11-09

US7816320

Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35

Hays, A.M.; Buechler; (+1)

2010-10-19

US7879580

Methods for high fidelity production of long nucleic acid molecules

Carr, P.A.; Chow, B.Y.; (+3)

2011-02-01

WO 2010/071602 A1

PCR-based method of synthesizing a nucleic acid molecule

WO 2010/132019 A1

Gene synthesis method

WO 2008/112683 A2

Gene synthesis by circular assembly amplification

WO 2008/112683 A3R4

Gene synthesis by circular assembly amplification

EP2190988A4

Integrated microfluidic device for gene synthesis

The Drawbacks of Past Inventions

The bottlenecks of gene synthesis lie in the high cost of
oligonucleotide synthesis, the constraint of the length of the
gene, the post-synthesis sequencing as well as the high error
rates. For example, the error rates ranged from 1/100 to
1/300 according to one document [18], when using phosphoramidite chemistries to produce conventional oligonucleotides. Since the errors might be deletions, substitutions
and sometimes insertions, this will consequently lead to
more sequencing in order to find an error-free clone, as it can
be predicted from a binomial distribution given an error rate
and gene length [19]. While the long synthetic DNA is in
high demand, a cheaper, easier, faster and more accurate

Daniel, G.G;

Venter J. C.;

Huang M.C.;
Li M.H.; (+2)
Li M.H.;
Jackie Y. Y.; (+2)
Duhee B.,
George M. C.
Duhee B.,
George M. C
Li M., Ying J.Y., Huang M., Pin
Ng, S.

2010-02-01

2009-08-20

2010-06-24

2010-11-18

2008-09-18

2008-09-18

2010-12-22

technique is badly needed to meet the needs of ordinary

laboratories and industry at large.
Multiplexing for Polymer Synthesis [5]
This method is created by Rinderknecht et al. Multiplexing was allowed in the synthesis of nucleic acids, which
means a large number of sub-reactions could occur in a relatively small size or volume, while it produced a relatively
long stretch of a polymer.
Many reaction sites such as reaction wells or sites was
fabricated to create short polymer fragments initially. A
monomer was attached to the surface of a reaction site, coupled to next monomer via light irradiation in the 100 to 1000

18 Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

nm wavelength range. The process was repeated until a

building fragment was constructed from scratch. This procedure was performed in parallel so many building fragments
were simultaneously synthesized, which were then removed
from reaction sites by irradiation with different wavelengths
from above, and subsequently used to synthesize a subpolymer. The reaction sites and combination site were fluidly connected. The subpolymers are stored in different storage sites, later combined to form a desired polymer. Ligations were also integrated into the process to produce polymers.
Sequential Addition of Short DNA Oligos in DNAPolymerase-based Synthesis Reactions [20]
This patent describes the use of a multiplicity of oligos to
assemble full length sequences. As genes synthesis premium
lies heavily in oligo synthesis cost, the use of tetramers can
greatly cut the cost down to negligible level. This is due to
the fact that the combinatorial complexity of tetramers is 256
(44), which can be assembled to form sequences of any size.
To avoid cross hybridization when using 2 base overlaps in
PCR, oligos 20 to 100 bases in size were initially constructed
using limited number of tetramers. Subsequently a second
PCR was performed to fabricate a full length gene. The oligos could be gradually added, thus temporarily separated,
eliminating incorrect hybridizations. Pentamers, hexamers,
etc. could be also used to give rise to different reading
frames. Larger oligos could be used for difficult regions having heterogeneous melting temperatures. The oligos were
preselected using computer softwares and bioinformatic approaches.
Methods for High Fidelity Production of Long Nucleic
Acid Molecules [21]
This patent provides solutions in addressing errors in the
synthesis of long DNA molecules. It employed a forcefeedback system using magnetic, or optical tweezers, or
both. DNA growing from a solid support was held in a fixed
equilibrium position, by applying an electric field and a
counteracted magnetic field. When an oligo annealed to the
growing DNA chain, and negative charges carried by phosphate groups were also added to the DNA chain, so a magnetic force was needed to counter the electrophoretic force
for keeping DNA in equilibrium position. With the help of
optical systems, the bean velocity and restoration force could
be used to ensure the length of the added strand to be correct.
Similarly, DNA growing from a fluorescent bead with a
functional phosphate group passed through a medium electrophoretically with excess ATP, kinase and ligase. Triphosphate formed on the bead changed its rate of motion, indicating a successful reaction. When a nucleotide was then ligated
with the DNA chain and a diphosphate was released, the rate
of bead motion was reduced.
Alternatively, an oligo interacting with a growing DNA
chain would melt away if there was a mismatch when temperature approaches the melting point. A drop in magnetophoretic force or increase in electrophoretic force to keep the
bean in position would signal that a mismatch was detected.

Yu et al.

A nanopore device was also adopted to monitor the deprotection of the 5 protecting groups to eliminate deletion
errors. In the single molecular synthesis system, the growing
chain could be shown to be deprotected when the wash was
flowed through the nanopore and fluorescence was detected.
The researchers also pointed out that a crosslinking between mismatch binding proteins with erroneous DNA
would prevent its amplification by DNA polymerases.
Methods for Generating Polynucleotides having Desired
Characteristics by Interative Selection and Recombination [22]
This patent describes the use of oligos in the conventional gene shuffling process. These oligos can have identity
to some regions and heterology to others. This provides unlimited diversity to the parental sequences for PCR shuffling
and assembly. Eventually affinity screening can be applied
to pull out positive clones.
Removal of Transcriptional Regulatory Sites and Gene
Expression [23]
This patent used gene synthesis to remove transcriptional
regulatory sites, including transcriptional binding sites, intron splicing sites, poly-adenylation sites, promoters and
enhancers, so the synthetic molecules had at least 3 fold less,
preferably 5 fold less regulatory sequences. Through a further optimization of codon usage via the use of favorite
codons, protein expression levels could be markedly enhanced.
Method for the Complete Chemical Synthesis and Assembly of Genes and Genomes [24]
It is a method designed by Glen A Evans for the complete chemical synthesis and assembly of genes and genomes. It provided an efficient way to generate nearly any
nucleic acid sequence. This approach is completely synthetic
by nature, so, there are no constraints, even for constructing
very long stretches of nucleic acid. Integrating gene parts
and functional elements via the use of simple computer
software, it is possible to construct a virtual polynucleotide
in a computer which is subsequently used as the blueprint for
DNA synthesis.
Different sequences are needed for transferring to different kinds of cells with corresponding origin of replication.
The genome sequence was broken down into a set of overlapping oligonucleotides of specified length by computer
software. To make sure a single mixture contained equal
concentrations of each, the concentrations of oligomers
needed to be balanced. Then the oligomers would be coannealed and a virtual gene or genome is constructed from
scratch. The DNA sequence was ligated via T4 DNA ligase
or topoisomerase 2, and then removed from the solid support
if attached. After a final ligation, the molecule would be
transferred into host cells.
Synthesis of Sequence-Verified Nucleic Acids [18]
The method documented by Stahler et al. could produce
synthetic nucleic acids from sequence-verified building

Recent Patents on Oligonucleotide Synthesis and Gene Synthesis

blocks, which reduced expense and efforts. This invention

provided an improved method for the quality control and the
subsequent preparation of DNA fragments in a high
throughput fashion.
The features of the invention are as follows: Initially,
sequences could be synthesized in situ with flanking short
sequences used as universal primers, which could be cleaved
by type II endonucleases. Oligos were assembled via PCR
amplifications, which could be done in micro-channels and
reaction vessels. Shortened fragments which impeded full
length gene assembly were diluted hence present at negligible concentration. Emulsion-based PCR could be conducted
to amplify clonal molecular populations or individual molecule from a mixture of DNA fragments, which could then be
subjected to next generation sequencing. Correct molecules
were subsequently retrieved and cloned, and eventually
characterized.
Integrated Microfluidic Device for Gene Synthesis [25]
Latterly, there was a demonstration of an integrated microfluidic device, which was able to perform two-step gene
synthesis in assembling a pool of oligonucleotides into genes
with the desired coding sequence. This invention has come
up with an integrated lab-on-a-chip microsystem to conduct
automatic gene assembly from short oligonucleotides.
The oligonucleotides were assembled into a DNA sequence for encoding genes and genomes based on known
assembly methods such as PCR or LCR. The fabricated device was combined with a miniaturized thermal cycler to
perform gene synthesis. Oligonucleotides were assembled
into genes by PCA, followed by a second PCR to generate
the full-length gene. Solid-phase purification from the PCR
reaction mixture was conducted for the synthesized gene.
Accordingly, one facet of the invention points it to a twostep method for synthesizing double-stranded DNA in a microfluidic device. Another facet identifies it as a one-step
method for synthesizing DNA duplex in a microfluidic device in combination with a purification step.
A More Efficient One-Step Gene Synthesis Method [26]
This method was established by Li et al. In the two-step
PCR protocol, amplification and assembly are performed
separately. While being efficient, it is a lot more costly and
laborious than the one-step process. Accordingly, this work
provided a method that integrated the advantages of the onestep process with the assembly efficiency of the two-step
process in gene synthesis. The amplification primers were
designed such that they had two distinct melting temperatures in order to minimize the competition between polymerase cycling assembly and PCR amplification in the onestep nucleic acid synthesis and to maximize the efficiency of
the full-length gene amplification.
The outer primers and inner oligonucleotides were designed with a substantial melting temperature difference. A
high annealing temperature corresponding to the Tm of oligonucleotides was used for the first 20 cycles aimed at assembly. This was followed by a low annealing temperature
corresponding to the Tm of outer primers to boost the fulllength amplification. The outer primers were prevented from

Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

19

annealing during the early assembly process when they were

subjected to an elevated annealing temperature, which excluded them from mis-pairing with other oligonucleotides
[27].
CURRENT AND FUTURE DEVELOPMENTS
The research in gene synthesis has gained momentum in
the last decade. Circular assembly amplification has been
designed to construct exonuclease-resistant circular DNA via
simultaneous ligation of oligonucleotides [28]. Its combination with subsequent mismatch targeted endonuclease
mediated degradation of the ligation mixture considerably
increased error free products. A highly repetitive DNA sequence has been assembled with this approach, showing
promise in resolving sequence homologies.
In 2009 and 2010, 2 studies were conducted by Gibson
et al. [29-30], who described an isothermal method for assembling overlapping DNA molecules by the combined actions of an exonuclease, a DNA polymerase and a DNA ligase in a single step effort. Single-stranded overhangs could
be generated by exonuclease, which were subsequently annealed, repaired and ligated. The 50C isothermal assembly
system was designed to take advantages of the activities of
the 5 T5 exonuclease, Phusion DNA polymerase manufactured by New England Biolabs and Taq DNA ligase, and
was able to construct fragments large or small.
Gibson et al. reported the synthesis of a 582, 970 base
pair Mycoplasma genitalium genome in 2008 [31]. This synthetic genome carries the complete genetic blueprint of wildtype M. genitalium G37 except a MG408 gene was inserted
with an antibiotic marker for selection. The DNA synthesis
was from scratch, albeit the primary cassettes were purchased from several commercial venders for time frame consideration. In a hierarchical approach, overlapping fragments
with sizes of 5 to 7 kilobases, were assembled by in vitro
recombination to yield transitional assemblies of approximately 24 kb, 72 kb, and 144 kb, which were then cloned in
bacterial artificial chromosomes and propagated in E. coli.
Although hampered by inability to propagate large size insert
clones in E. coli, researchers eventually succeeded in synthesizing the complete genome in Saccharomyces cerevisiae via
transformation associated recombination. In the in vitro recombination experiments, the exonuclease activity in the T4
polymerase in the absence of dNTPs was used to generate
recessed ends. The annealed fragments were extended and
ligated using Taq polymerase and Taq ligase at 45C in the
presence of dNTPs.
Subsequently a 1.08megabase pair Mycoplasma mycoides synthetic genome, dubbed JCVI-syn1.0, was synthesized and transplanted into a M. capricolum recipient cell to
get booted [32]. The new cells, with 14 non-essential genes
deleted in the genome and 4 watermarks inserted as well as
numerous other modifications, had expected phenotypes and
were capable of sustained growth. This ushered a new era in
synthetic biology and artificial life, made possible by 15
years of efforts to turn idea on the drawing board into science facts.
The column based synthesis of oligonucleotides is both
costly and low throughput, creating bottlenecks in gene syn-

20 Recent Patents on DNA & Gene Sequences 2012, Vol. 6, No. 1

Yu et al.

thesis. Oligonucleotides from DNA microchips could cut

costs over ten fold [33-35]. However, the high error rates and
cross hybridizations of the oligonucleotide mixtures have
overshadowed previous efforts on microchips. Using highfidelity DNA microchips, integrated with selective oligonucleotide sub-pool amplification using common primers followed by cleavage with type IIS restriction enzymes, streamlined gene assembly protocols and error correction, Kosuri
et al. came up with a approach for highly parallel gene
synthesis [36]. This method has been validated by
assembling 47 genes, including 42 therapeutic antibody
sequences having about 61% homologies. These genes were
assembled from a mixture containing 13, 000
oligonucleotides encoding about 2.5 mega bases of DNA.
This method has the potential of cutting down the cost of
gene synthesis by orders of magnitude.
A single microchip, integrating the synthesis of oligonucleotides using inkjet printing, isothermal oligonucleotide
amplification and parallel gene assembly, and a mismatchtargeted endonuclease for error correction, has been reported
Table 4.

Technologies

Challenges Ahead

Genome synthesis and engineering

Prompt editing may be required in the genome synthesis process to avoid delays

Metabolic engineering

Regulatory elements need to be fine tuned

Post synthesis error correction

Integration into microchips or robotic workstation is an advantage

Parallel synthesis on microchips

Difficulties are present for skewed GC or AT content

Sequences of extraordinary GC or AT content

Novel technologies may be required for rapid synthesis

Repetitive sequences

Parallel synthesis may be difficult

Artificial or chimeric proteins

Efficient screening assays need to be envisaged as the odds for successful isolation of artificial proteins could be very low

This work was supported by Guangdong Science and

Technology Program (No. 2008B020100001), Guangdong
Natural Science Foundation of China (S2011010004264) to
Q. Liu, Laboratory Open Fund of Sun Yat-sen University to
C. Yu and Q. Liu (KF201028), Undergraduate Research
Fund at SYSU to T. Yu, and Innovation Program of Featured
Specialty Development Fund in SYSU to W. Piao. We thank
anonymous reviewers for valuable suggestions, and Yan Shi
for proofreading.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
REFERENCE

[2]

Many researchers are now engaged in genome synthesizing, metabolic engineering, etc. (Table 4). Since errors could
be introduced to synthetic genomes, consequently editing is
required. Completely synthetic genomes are still some distance away. One future challenge will be the synthesis of
DNA with extremely skewed GC or AT contents which may
serve as functional elements in natural or synthetic genomes.
With the advances in integrated microchip technology, and
the boom of commercial venders, gene synthesis cost is
bound to plummet. Synthetic genomes or engineered metabolic pathways may be one day exploited to produce biofuels at industrial scale, and enable the manufacture of many
drugs, vaccines or valuable products.

Technologies to Watch and Challenges

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