Molecular Cloning

Handbook
Past, Present, and Future
Techniques of Molecular Cloning

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troubleshooting guide
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Table of Contents
Traditional Molecular Cloning Overview
The History of Molecular Cloning 1
Looking for an ORF or mutant ORF clone? Molecular Cloning Strategies 3
GenEZTM Clones make ORFs more accessible and more customizable than ever. Traditional Molecular Cloning Bottlenecks 5
Molecular Cloning Troubleshooting Guide 7
Search for your ORF using our ORF clone database, select your vector, and place your order online. GenEZTM
ORF Clones are delivered in our standard mammalian expression/transfection-ready vector, or can be Other Limitations of Traditional Molecular Cloning 11
subcloned into the vector of your choice and start at just $99/clone. GenEZTM clones are shipped in as few as
5 business days. Gene Synthesis – The Solution to the Limitations of Traditional Cloning 13

How Gene Synthesis Revolutionized Molecular Cloning

What is Gene Synthesis? 14
What Can Be Synthesized? 16
How is Gene Synthesis Performed? 17
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References 30

Quotations and Ordering:

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Traditional Molecular Cloning Overview cutting and gluing of DNA molecules together. Later innovations such as
transformation, PCR, Sanger sequencing, and more recently, gene synthesis would
The History of Molecular Cloning make molecular cloning one of the most prolific tools of the molecular biology
laboratory.

The key principles of molecular cloning were discovered a little over 50 years ago.
Since then, molecular cloning has become one of the most powerful tools of the Figure 1. Timeline of molecular cloning history
molecular biology laboratory enabling the expression of the smallest genes, as well as
the engineering of whole genomes.
1952-1953 1977
Genetic basis for phage restriction described Sanger DNA sequencing & the
The advent of molecular cloning was spawned from a number of observations that first cloning vector, pBR322,
centered around DNA recombination, namely the exchange of DNA bewteen developed
bacterial and bacteriophage genomes [1, 2]. Key to these pioneering observations 1962 1967 1972
Model of DNA DNA ligases First
was the discovery that bacteriophage λ formed a circle when it entered the host recombination in isolated & transformation of
bacterial cell. In particular, phage DNA was observed to have single stranded DNA phages published characterized E. coli
flanking each end. The ends were complementary to each other and were called
cohesive sites or “cos sites”. The cos sites could reanneal to make the phage DNA
circular. Then, excision and insertion events led to the incorporation of bacteriophage
DNA into the host genome [3]. Campbell’s model of λ prophage excision and insertion
led to the classic principle of DNA recombination. In particular, today’s “sticky ends”,
formed by DNA digestion using restriction enzymes mimic the functionality of cos
sites allowing for the insertion of a target sequence into its destination vector.

Next came the discovery of another key tool in molecular cloning, the restriction
enzyme. It was discovered that methylation of phage DNA by host methyltransferases
prevented phage DNA from being destroyed by host enzymes called restriction
nucleases. Foreign DNA molecules not having the methylation patterns in accordance 1983
1976 PCR conceived
with their host (or that were unmethlyated) were recognized as foreign and Synthesis of of and
destroyed by host restriction nucleases [4, 5]. The first restriction nucleases were first synthetic demonstrated
characterized by Meselson and Yuan [6]. Then, Kelly and Smith showed that 1961 1970
gene
restriction enzymes recognize and cleave specific nucleotide sequences [7]. Today, First restriction nucleases isolated Sequence specificity of restriction nucleases
restriction enzymes are used as biological scissors to cut out the precise DNA desired demonstrated
to create recombinant sequences. The Escherichia coli strains of today used for
transformation of cloned recombinant DNA lack these enzymes.

Finally, came the discovery of enzymes called ligases, which could join two DNA
molecules together [8-12]. The discovery of ligases solidified the groundwork for the
basic principles of molecular cloning – the creation of recombinant DNA via the

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Molecular Cloning Strategies transformants will contain the gene in the sense direction and half will contain the
gene in the antisense direction. However, cells transformed with toxic genes may all
Since the seminal discoveries of the basic principles underlying molecular cloning, a display the genes in the antisense direction, since cells containing the sense directed
number of cloning strategies have been developed to improve on the ease and speed genes will not survive. In addition, survivor cells containing toxic genes oriented in
at which DNA fragments can be recombined. the sense direction may be mutated to encode a less toxic protein.

Traditional cloning Figure 3: Schematic of TA cloning

Traditional cloning, also called PCR cloning, requires the use of the polymerase chain A A
1 T T 2 3 4 5 6
reaction (PCR) to amplify the template sequence of interest (usually the gene of
interest) and add restriction sites to the ends of the sequence. Restriction enzymes
are used to cut both the template of interest and the target vector, and DNA ligase is
Gene of interest is Ligation of Transformation Plasmid isolation Construct
used to join the sticky ends of the template and vector together. Traditional cloning amplified by PCR gene and verification by
and contains vector sequencing
allows for flexible DNA sequence manipulation, which facilitates the building of nearly adenosine
any desired construct. However, the checkpoints and optimization procedures overhangs. Plasmid
contains thymine
required for traditional cloning can be cumbersome, and the reagents required can be overhangs.
expensive.
Seamless cloning
Figure 2: Schematic of PCR cloning
Seamless cloning technologies eliminate the requirement for restriction enzymes.
MCS
This can be advantageous when an insert contains a number of restriction sites
1 2 3 4 5 6 7 within its sequence, making it difficult to identify restriction enzymes that will not cut
P

P
the gene of interest during the cloning procedure. Seamless cloning takes advantage
of homologous recombination and there are numerous variations on the technique.
Ligation of Transformation Plasmld isolation Construct In general, the procedure consists of adding flanking sequences approximately 15 bp
Gene of interest verification
is amplified by digestion by in length to both the insert and vector via PCR. Exonucleases are used to chew back
PCR. Plasmld products sequencing
contains multiple the insert and vector sequences and the DNA is joined using recombinase enzymes
cloning site, or DNA ligase. Seamless cloning has been simplified by the development of kits that
encompassing Digestion of PCR
multiple product and vector by already contain the target vector and a proprietary mix of enzymes required for the
restriction restriction enzymes.
enzyme Dephosphorylation of recombination reaction. For instance, GenScript’s CloneEZTM Kit can clone inserts up
recognition sites. vector may be required.
to 10 kb in 30 minutes, and can also be used for high-throughput cloning projects.

TA cloning
Figure 4: Schematic of seamless cloning
TA cloning is one of the simplest forms of cloning. In this method, vectors containing
5' thymine overhangs are used to accept PCR products in which additional 3' 1 2 3 4 5 6
adenosine overhangs have been added on by the nature of TAQ polymerase
amplification. TA cloning has the advantage of ease and speed, since no restriction
digestion step is required. In addition, TA cloning kits contain reaction buffers that PCR Exonucleases Transformation Plasmld isolation Construct
amplification of chew back insert verification
contain the pre-mixed vector, ligase, and buffer, cutting ligation reaction time to as gene of interest. and vector by
few as 5 minutes. The disadvantage of TA cloning technology is that the cloning is not Addition of 15 sequences. sequencing
bases flanking Joining of vector
directional, meaning the gene of interest may be inserted into the target vector in sequence and insert by
homologous to recombinase or
either the sense or antisense orientation. Normally, half of the subsequent vector. ligase.

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 may require an additional de-phosphorylation reaction step or the insert may require
Traditional Molecular Cloning Bottlenecks an extra phosphorylation step, to prevent vector re-annealing and improve
insert-vector ligation efficiency, respectively. In addition, solvent contaminants from
Technical bottlenecks of traditional molecular cloning can cause the process to be
DNA purification procedures such as agarose gel extraction can lower ligation
time consuming and expensive. Smoothly run cloning experiments can take about
efficiency.
2-3 weeks to complete. However, many cloning experiments require troubleshooting
in at least one phase of the process, increasing both cost and time consumption.
Mutations – Mutations can be introduced into the molecular cloning process during
Below is a list of common bottlenecks that plague the traditional cloning process.
PCR amplification or plasmid propagation. Typical Taq DNA polymerases do not
contain proofreading subunits. Though high-fidelity Taq DNA polymerases that
Unstable, low yield RNA extraction – Molecular cloning often begins with total RNA
contain proofreading subunits, such as Pfu, have been developed, reaction conditions
extraction from the host containing the gene of interest. For the cloning of protein
such as sustained high temperatures can still cause DNA damage that results in
coding sequences, mRNA extraction is required. mRNA copy numbers for some
mutations.
genes can be low, making it difficult to amplify the sequence via RT-PCR. In addition,
RNA extraction from some host tissues may prove difficult due to the harsh
During plasmid propagation, toxic genes may be expressed at low levels due to RNA
conditions under which some cell types must be lysed. Finally RNA is inherently less
polymerase read through of antibiotic resistance genes, or leaky promoters. Proteins
stable than DNA, and great precautions must be taken to prevent the action of
or enzymes encoded by toxic genes may cause the transformed cells to die, resulting
RNases and the degradation of RNA by repetitive freezing and thawing.
in only the survival of transformants containing mutated genes encoding less potent
versions of the target protein or enzyme.
Difficult PCR program optimization – Following the synthesis of cDNA from mRNA
molecule templates, a PCR program must be designed to amplify the gene of
interest, as well as add additional elements such as restriction sites or
detection/purification tags. Intrinsic properties of gene sequences such as high GC Figure 5: Traditional molecular cloning steps and timeline
content, long stretches of the same polynucleotide, and sequences encoding hairpin At best, traditional cloning may take only 2 weeks, but troubleshooting after
loop structures can all hinder PCR efficiency. each phase may extend the cloning process to multiple weeks or months.

DNA losses from purification steps – DNA must be purified at numerous steps in the
Day 4 Day 8
traditional cloning process. PCR products must be purified from reaction 1. Purify digested products 1. Receive plasmid
components; digested genes and vectors must be purified from restriction enzymes, 2. Set up ligation reaction sequence
buffers, and digestion products; and plasmid DNA must be extracted and purified Day 1 Day 6
from cellular material. At each step, DNA recovery % can vary from as high as 95% to Order primers, prepare source 1. Verify positive colonies via PCR
of template 2. Set up overnight culture
as low as 60% depending on the purification method and quality of the DNA. Low
recovery percentages or contamination during the purification procedure can result
in DNA losses so great that the next step of cloning cannot be carried out, requiring 1 2 3 4 5 6 7 8
steps such as PCR, digestion, or transformation to be repeated.
Day 5
Low ligation efficiencies – Following the digestion of the gene insert and the target Transform E. coli
Day 3
vector, a ligation reaction is performed to join the two molecules. The efficiency of 1. Troubleshoot & optimize PCR Day 7
ligation reactions are dependent on a number of variables including vector to insert 2. Purify PCR products 1. Perform plasmid extraction
3. Digest PCR products & plasmid 2. Sequence plasmid
ratio and salt concentration. If the ratio is too low, or plasmid digestion is
incomplete, excess vector may re-anneal without the insert. In some cases the vector

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7
Molecular Cloning Troubleshooting Guide
Problem Potential Causes Solutions

Number of PCR cycles is insufficient • Increase number of PCR cycles by 5.

Template is degraded • Use electrophoresis to check DNA quality.

• Check DNA ratio of absorbance at 260 and 280 nm. Pure DNA

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should have a 260/280 ratio of ≥ 1.8.
Template is contaminated with PC
• Use less volume of the template in the reaction.
inhibitors
• Use DNA clean-up kit or ethanol precipitation to remove
contaminants.

• Follow general rules of PCR design: Annealing temperature =

Thermocycler program annealing and
lowest primer Tm - 5 °C, Extension temperature = 72 °C.
extension temperatures are not
• Decrease annealing temperature by 6 to 10°C in stepwise
optimal
Low or no PCR fashion.
product yield
Reaction is missing Taq polymerase or
• Make sure each component was added to PCR reaction.
other reaction component

• Check primer concentration; increase concentration if

Primer concentration too low
necessary.

Target sequence is not in DNA • Re-extract DNA from source.

template • Test another region of template.

Not enough template • Increase concentration of DNA template.

Reaction component concentrations • Check recommended primer concentrations (normally from

not optimal 0.05–1 mM) and Mg++ concentrations (normally from 0.2–1 mM).

• Check expiration date of components.

Reaction mix components are • Aliquot biological components of reaction mixture and avoid
compromised multiple freeze-thaw cycles.

• Follow general rules of primer design: Length from 18-30

nucleotides, GC content from 40-60%, Tm of primers within 5°C
Primer design not optimal (causing of each other.
non-specific annealing, or primer • Avoid stretches of 4 or more of the same nucleotide or
dimer formation) dinucleotide repeats.
• Avoid self-complementary sequences within primers.

• Follow general rules of primer design: Length from 18-30

nucleotides, GC content from 40-60%, Tm of primers within 5°C
Primer design not optimal (causing of each other.
non-specific annealing, or primer • Avoid stretches of 4 or more of the same nucleotide or
dimer formation) dinucleotide repeats.
• Avoid self-complementary sequences within primers.
Multiple/non-
specific • Re-extract template.
Template or reaction mixture • Try new reaction mixture.
products from
components are contaminated • Use filter pipette tips and wear gloves during reaction set-up.
PCR reaction

Annealing temperature too low • Incrementally increase annealing temperature.

Primer concentration too high • Use less primer.

• For plasmids use 1 pg–10 ng of DNA / 50 μl reaction.

Template concentration is not optimal • For genomic DNA use 1 ng–1 μg of DNA / 50 μl reaction.

• Check enzyme expiration date.

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Enzyme quality is compromised • Avoid multiple freeze thaw cycles of enzyme.

DNA from a bacterial source or eukaryotic source may be blocked

Methylation of template sequence by Dam and Dcm methylation or CpG methylation, respectively.
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prevents enzyme from cutting • Use a Dam- or Dcm- strain.

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Not enough incubation time • Add 1 to 2 hours to incubation time.

Inhibitor contamination can originate from miniprep kits, or leftover

PCR reaction components:
Incomplete or Template/plasmid contamination with • Use dialysis or spin column to remove contamination.
no inhibitors • Dilute or use less volume of DNA - DNA solution should compose
template/vector no more than 25% of digestion reaction.
digestion • Use PCR clean up kit to remove PCR reaction components.

Enzyme concentration too low • Use 3–5 units of enzyme / μg of DNA.

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Incompatible buffer was used • Use recommended buffer with enzyme.

• Use commercial kit to clean up template.

Template is contaminated with
• Use running buffer and agarose gel made with fresh nuclease-free
Digestion nucleases
water.
product is
smeared on gel • Decrease units of enzyme.
Enzyme is bound to DNA substrate • Remove enzyme from DNA by adding 0.1–0.5% SDS to loading
buffer.

• Use minimum incubation time.

Template/vector
Star activity (enzyme cleaved similar • Reduce number of enzyme units in reaction.
appears to be
recognition sequence) • Use compatible buffer with enzyme.
over digested
• Use a high-fidelity enzyme.

• Ensure the correct antibiotic was applied to plates.

Wrong antibiotic was used or antibiotic • Use only concentration recommended by competent cell or
concentration was too high antibiotic manufacturer.

• Thaw competent cells on ice and use immediately.

• Check expiration date of cells.
Competent cell viability is low • Do not re-freeze cells.
• Do not vortex cells - gently tap to mix.

DNA insert encodes protein that is • Use a lower incubation temperature (25 – 30°C).
toxic to cells • Use a cell strain and vector designed for tightly controlled transcription.
Few or no Heat-shock incubation too long • Reduce incubation time from 45 to 25 seconds.
colony
transformants Construct is too big • Use electroporation for vectors over 10 kb.

Too much ligation mixture was used for Ligation reaction components can inhibit transformation.
the transformation • Dilute ligation reaction with TE buffer (up to 5 times).

• Use no more than 1-10 ng of DNA in 5 μl for a 100 μl reaction or in

Too much DNA in reaction
1-3 μl for a 50 μl reaction.

• Vector insert ratio not optimal. Use a vector:insert molar ratio

Low ligation efficiency from 1:1 to 1:10.
• Use a DNA concentration of 1-10 μg/ml.

Construct recombined with genomic DNA • Switch to a Rec A- cell strain.

Antibiotic concentration too low • Use antibiotic concentration recommended by manufacturer.

No plasmid in
colony
• Aliquot working volumes of antibiotic and avoid freeze-thaw cycles.
transformants Antibiotic is degraded
• Add antibiotic to liquid plate media after sufficient cooling.

No insert in colony • Vector insert ratio not optimal. Use a vector:insert molar ratio
transformant Vector re-ligation from 1:1 to 1:10. Use a DNA concentration of 1-10 μg/ml.
plasmids • Dephosphorylate DNA with phosphatase to prevent re-ligation.

DNA insert encodes protein that is • Use a lower incubation temperature (25 – 30°C).
Sequencing of
toxic to cells • Use a cell strain and vector designed for tightly controlled transcription.
transformant
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plasmid reveals
Mutations introduced by initial PCR • Use a high-fidelity polymerase.
wrong plasmid
sequence
Inconclusive sequencing artifacts • Repeat sequencing reaction.
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Other Limitations of Traditional Molecular Figure 6: DNA sequence features that influence protein expression levels

Cloning 2. mRNA processing and stability Deviant folding

DNA
cryptic splice sites
In addition to the bottlenecks encountered in traditional molecular cloning, other
mRNA secondary structure
limitations avail. Traditional cloning is most useful for stitching together naturally 4
Natural folding

stable free energy of mRNA

occurring sequences. Engineering optimized or non-natural sequences can be much
more difficult. In particular, gene sequences may be engineered to contain mutations 2 4. Protein folding
for performing functional assays of the resulting protein or for characterization of the codon context
disease state of a protein. In other cases, a researcher may desire to optimize the codon-anticodon
interaction
codons of a natural gene sequence to enhance protein expression. In both cases, translation pause
CGU
additional manipulation of the cloned gene is required, which may result in 1 sites
additional DNA losses and unwanted mutations. 3
1. Transcription
cis-regulatory elements (TATA box, 3. Translation
termination signal, protein binding codon usage bias
Codon Optimization sites, etc.) ribosomal binding sites (e.g. IRES)
In order to characterize a gene’s function or purify a protein for structural study, chi sites premature polyA sites
polymerase slippage sites
researchers frequently need to express a gene in a host cell or model organism
different from the one in which it appears in nature. Unfortunately, heterologously
expressed genes often suffer from low rates of protein expression. A common cause
Modifying a DNA sequence to optimize it for efficient heterologous protein
of low expression levels is the variation in codon usage frequency between different
expression while preserving the amino acid sequence is called codon optimization. In
species. This can cause the translation of heterologously expressed genes to stall due
a study by Burgess-Brown et al. [13] that investigated the expression of 30 human
to tRNA scarcity, leading to lower protein levels and increased rates of improper
genes in E. coli, codon optimization increased both the total protein yield and the
protein folding. The degeneracy of the genetic code makes it possible to change the
protein solubility, compared to heterologous expression of native gene sequences.
DNA sequence in a way that does not alter the final amino acid sequence, but does Codon optimization algorithms have improved since this 2008 study, and codon
have significant effects on the efficiency of transcription, mRNA stability, translation, optimization is now widely used by researchers to facilitate the purification of
and protein folding. Aside from codon usage bias, many other features of a DNA proteins for structural studies, enzyme kinetics, and other biochemical investigations.
sequence affect the efficiency of transcription, the proper splicing and processing of
mRNA, and mRNA stability, all of which in turn reduce the ultimate protein yield
(Figure 6). Mutagenesis
The mutation of gene sequences is useful for identifying amino acids crucial to
protein function, studying disease states of native proteins, facilitating drug discovery
or developing new protein variants (mutants) with properties desired for medical,
industrial, agricultural, or other applications. Traditional methods for inducing
random mutations include subjecting live cells or animals to UV radiation or chemical
mutagens in order to induce DNA damage. DNA repair enzymes can, in some cases,
introduce or preserve mutations resulting from damage such as single- or double
strand breaks, but this method leads to high rates of lethality and low rates of useful
mutant phenotypes.

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Depending on the type of mutagen used, mutations may be biased, limiting the
range of possible mutants to only a fraction of those that are theoretically possible
How Gene Synthesis Has Revolutionized
or that can be obtained through other strategies. Molecular Cloning
Site-directed mutagenesis may be designed based on experimental data such as
X-ray crystallography data that identifies residues of interest. Site-directed What is Gene Synthesis?
mutagenesis can be performed using PCR-based methods with mutated primer
sequences to create and selectively amplify mutated sequences. This type of Gene synthesis technologies have revolutionized biology research and provide a more
mutagenesis must be followed by sequence verification, and can be tedious, straightforward, faster, and less costly alternative to traditional molecular cloning.
error-prone, and costly. Gene synthesis involves the de novo chemical synthesis of DNA, differing from
traditional molecular cloning in that no template is required. Gene synthesis allows
researchers to specify a desired sequence (native or engineered) and custom-build it
directly.
Gene Synthesis – The Solution to Gene synthesis refers to chemically synthesizing a strand of DNA base-by-base. Unlike
the Limitations of Traditional Cloning DNA replication that occurs in cells or by PCR, gene synthesis does not require a
template strand. Rather, gene synthesis involves the step-wise addition of nucleotides
Luckily, a method that circumvents the limitations of traditional molecular cloning
to a single-stranded molecule, which then serves as a template for creation of a
strategies has been developed – gene synthesis. Gene synthesis is the de novo
complementary strand.
synthesis of a DNA strand. No template is required for gene synthesis and nearly any
sequence that can be designed can be synthesized. In the next section the Any DNA sequence can be synthesized, including sequences that do not exist in
applications and techniques that demonstrate how the versatility of gene synthesis nature, or variants on naturally-occurring sequences that would be tedious to
has both accelerated and revolutionized molecular cloning are discussed. produce through site-directed mutagenesis, such as codon-optimized sequences for
increased heterologous protein expression. Synthetic DNA can be cloned into
expression vectors and used in any protocol that requires natural or recombinant
DNA. Synthetic genes are used to study all the diverse biological roles that nucleic
acids play, from encoding proteins and regulating gene expression in the nucleus, to
mediating cell-cell communication and building biofilms from extracellular DNA.

Due to gene synthesis, scientists are no longer limited to classical methods of

manipulating a single gene at a time; they now have the power to design or
reprogram entire genomes and cells which can advance the fields of healthcare,
agriculture, energy, and other fields of human endeavor. For instance, newly
identified viral genomes can be quickly synthesized to accelerate vaccine
development. Novel enzymes can be engineered to fight cancer and produce
sustainable biofuels. Genes can be engineered to enhance crop yields and reduce
vulnerability to the common pests and plant diseases that endanger food supplies
and contribute to global hunger. Designer metabolic circuits using interchangeable
synthetic parts can be built to create synthetic genomes and artificial cells for
studying the basic requirements of life.

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14
Methods for de novo chemical synthesis of DNA have been refined over the past 60 Figure 7: Timeline of gene synthesis technology development
years. Synthetic short oligonucleotides (oligos) serve as custom primers and probes
for a wide variety of applications. Longer sequences that serve as genes or even 2004
whole genomes can be synthesized as well; these sequences are typically produced 1950 Microchip- and
DNA polymerase isolated from E. coli. Arthur microfluidics-based methods
by synthesizing 40-200 bp oligos and then assembling them in the proper order. Kornberg wins 1957 Nobel Prize. for high-throughput gene
Many methods for oligo assembly have been developed that rely upon a DNA
synthesis are first published.
polymerase enzyme for PCR-based amplification, a DNA ligase enzyme for ligation of 1967 1990s
oligos, or enzymes that mediate homologous recombination in vitro or in vivo. Most DNA ligase is Improved
isolated and 1983 assembly methods 2011
sequences up to 1000 base pairs (1 kb) can be assembled in a standard molecular characterized by PCR is invented. allow synthesis of Sc2.0 project
biology lab, and commercial gene synthesis providers routinely synthesize sequences five independent Kary Mullis wins increasingly long launched to build
1993 Nobel Prize. genes. the first synthetic
over 10 kb. laboratories. eukaryotic genome.

The history of gene synthesis began in 1955, when Sir Alexander Todd published a
chemical method for creating a phosphate link between two thymidine nucleosides,
effectively describing the first artificial synthesis of a DNA molecule [14] The first
successful synthesis of an entire gene was reported by Gobind Khorana’s group in
1970; the 77 bp DNA fragment took 5 years to synthesize [15] Subsequent
1989
improvements in DNA synthesis, sequencing, amplification, and automation have 1983 First automated
made it possible now to synthesize genes over 1 kb in just a few days, and to Caruthers and oligonucleotide
2008
Matteucci invent First
synthesize much longer sequences including entire genomes. Gene synthesis can synthesizer machines
phosphoramidite complete
become commercially
now be easily and cost-effectively outsourced to commercial providers. GenScript, a DNA synthesis. synthesis
available. 2003 of a
pioneer in gene synthesis, was founded in 2002 and is the largest gene synthesis 1970 First bacterial
supplier in the world. First synthesis of an entire gene, a 77 bp yeast synthesis genome.
tRNA, by Gobind Khorana's group. of an entire
1955 viral genome,
First DNA molecule synthesized when two thymidine nucleosides that of phiX174
are joined by a phoshate link. Sir Alexander Todd wins 1957 bacteriophage.
Nobel Prize.

What Can Be Synthesized?

Gene synthesis can generate recombinant, mutated, or completely novel DNA
sequences without a template, simplifying the creation of DNA tools that would be
laborious to produce through traditional molecular cloning techniques. A wide
variety of types of sequences can be produced to aid in diverse research applications
(See Table 1). In addition to DNA sequences, RNA and oligos containing modified
bases or chimeric DNA-RNA backbones can also be synthesized. However, the most
widely used synthetic sequences are customized DNA of the following types:

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Table 1: Types of synthetic DNA and their research applications Figure 8: Steps in the de novo gene synthesis process

Types of Synthetic DNA Research Applications

cDNA / ORFs Over-expression or heterologous expression DNA Sequence Sequence Optimization Oligo Synthesis Gene Assembly
Selection and Oligo Design

Customized Expressing fusion proteins; high-level protein

coding expression from codon-optimized sequences for
sequences purification for enzymatic or structural studies

Monitoring gene expression downstream of Transformation, Quality Control:

Promoter-reporter Cloning into
manipulations to transcription factors, Transfection, Downstream Sequence Verification Vector of Choice
constructs signaling cascades, etc.
Application and Error Correction

Creating synthetic genes or genomes;

Genomic DNA studying gene structure, regulation, and
evolution. Step 1: Sequence Optimization and Oligo Design
Confirming function (promoter-bashing;
Mutant
amino acid substitutions); Protein engineering
sequences Sequence Optimization
(rational design or screening/directed evolution)
Once a gene of interest has been selected, the sequence to be synthesized must be
RNAi constructs Regulating (suppressing) gene expression;
(shRNA, miRNA, siRNA) intercellular communication
designed with the end application in mind. For example, codon optimization is
appropriate if the goal is to maximize heterologous protein expression levels,
Biofilm formation, intercellular signaling as in however, it may not be appropriate for the study of endogenous regulation of gene
Extracellular DNA
cancer metastasis
expression. For constructs containing multiple segments, the intended reading frame
should be maintained throughout the entire coding region. The addition of short
flanking sequences can facilitate later excision or recombination, via restriction
enzymes or similar tools. Sequences should be designed to minimize restriction
enzyme recognition sites or other sequences that might interfere with downstream
How is Gene Synthesis Performed? workflow. Careful attention should be paid to functional domains such as
cis-regulatory elements or RNase splice sites, which may be unintentionally
The basic steps of gene synthesis are: introduced during codon optimization. The presence of biologically functional
1. sequence optimization and oligo design sequences such as cis-regulatory elements or RNase splice sites on oligos may hinder
2. oligo synthesis in vivo assembly, maintenance, or expression.
3. gene assembly
4. sequence verification and error correction Some sequence features make synthesis more challenging, including: extremely
5. preparation of synthetic DNA for downstream applications high or low GC content; highly repetitive sequences; complex secondary structures
such as hairpins; unstable structural elements; polyA stretches; and longer
sequences, especially over 1 kb. These are all important features to consider when
selecting and optimizing sequences to synthesize, and they will increase the cost
and turnaround time of synthesis, whether it is performed in-house or outsourced
to a commercial service provider.

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Oligo Design
Step 2: Oligo Synthesis
After finalizing the sequence that will be synthesized, sequence analysis is required
to determine the best way to divide the whole gene into fragments that will be
synthesized and then assembled. Very large synthetic gene sequences, should be All DNA fabrication today begins with the step-wise addition of nucleotide monomers
divided into chunks of 500-1000 kb to be synthesized separately and assembled later. via phosphoramidite chemistry to form short oligonucleotides. Oligo synthesis via
phosphoramidite chemistry uses modified nucleotides, called phosphoramidites, to
While synthesis platforms utilizing phosphoramidite chemistry have very low error ensure that nucleotides assemble in the correct way and to prevent the growing
rates, errors do accumulate as strand length increases; therefore, gene synthesis strand from engaging in undesired reactions during synthesis. The phosphoramidite
typically uses oligos with lengths of 40-200 bp. The optimal oligo length depends group is attached to the 3' O and contains both a methylated phosphite and a
upon the assembly method that will be used, the complexity of the sequence, and protective di-isopropylamine to prevent unwanted branching. Phosphite is used
the researcher’s preferences. As a general rule, shorter oligos may have a lower because it reacts faster than phosphate. Methyl groups are attached to the
error rate, but will be more expensive to synthesize because more overlaps will be phosphite, and amino-protecting groups are added to the bases, to protect against
required. Longer overlaps increase the likelihood of correct assembly by decreasing unwanted reactions until oligonucleotide synthesis is complete. Although DNA
the rate of nonspecific annealing. Oligos should be adjusted for even lengths and synthesis in living cells always occurs in the 5' to 3' direction, phosphoramidite
equal melting temperatures. Each oligo design tool has its own default parameters; synthesis proceeds in the 3' to 5' direction. The first monomer is attached via the 3' O
for example, GeneDesign uses default settings of 60 bp oligos with 20 bp overlaps- to a solid support such as a glass bead, and its 5' O is initially protected from
values which typically work well with yeast and mammalian sequences which are nonspecific reactions by conjugation of a dimethyloxytrityl (DMT) group.
~40% GC.
Figure 9: Phosphoramidite reaction cycle
In addition to oligo length, some factors to
consider in selecting oligo sequences include GenScript’s proprietary Terminal,
Protected Monomer
DSOptTM Sequence Analysis
GC content, sequence repeats, and the
tool, developed through
tendency for hairpin formation. GC content years of experience as the
determines the stability of DNA strands and volume leader in gene
thus the melting temperature. All in vitro synthesis, takes the Repeat Steps 1-4
until chain is Step 1:
assembly methods, both ligase and guesswork out of selecting complete
Deprotection

polymerase based, rely upon the the best assembly method

and oligo design strategy
melting and annealing of oligos and thus
require that oligos have equal melting
for each project.
Step 4:
Phosphoramidite
Reaction Cycle
Terminal,
De-protected
Monomer
+ Activated
Monomer
temperatures. Oxidation

Repeated sequences may pose challenges for correct annealing or ligation of oligos Step 2:
Coupling
during assembly. Repeats may occur in many forms, including direct, inverted, Step 3:
palindromic, or tandem repeats. Improper hybridization or intramolecular binding Capping

such as hairpin structure formation can be avoided through careful design of oligo
sequences, including the overhangs or primers used to assemble them.
Unreacted
terminal
Monomers
are acetylated

19 www.genscript.com www.genscript.com 20
In Step 1: Deprotection, DMT is removed by washing with a mild acid such as vector [14]. Synthetic oligos are prepared for SLIC by PCR extension to introduce
trichloroacetic acid, exposing the 5' O for reaction. The second nucleotide is flanking regions of sequence homology. This facilitates recombination of fragments
introduced to the reaction with its own 5' O protected with DMT, while its 3' O is without any sequence restrictions or the introduction of restriction enzyme sites that
activated by the conjugated phosphoramidite group. Step 2: Coupling occurs when will produce permanent seams. The exonuclease activity of T4 DNA polymerase
the 3' O of the second nucleotide forms a phosphate triester bond with the 5' O of generates single-stranded DNA overhangs in the insert and vector sequences.
the first nucleotide. Step 3: Capping involves acetylating the 5' OH of any unreacted Homologous regions are annealed in vitro and undergo gap repair after
nucleotides to prevent later growth of an incorrect sequence. Acetic anhydride and transformation into E. coli.
dimethylaminopiridine are typically added to acetylate any unreacted terminal 5' OH
groups, but will have no effect on terminal nucleotides that are protected by DMT.
Step 4: Oxidation occurs upon the addition of iodine to convert the phosphate Step 4: Sequence Verification and Error Correction
triester bond into the phosphodiester bond that forms the
familiar backbone of DNA.
Due to the inherent potential for error in each step of gene synthesis, all synthetic
Once the desired chain is complete, all of the protecting groups must be removed, sequences should be verified before use. Sequences harboring mutations must be
and the 5' end of the oligo is phosphorylated. Prematurely terminated strands can be identified and removed from the pool or corrected. Internal insertions and deletions,
removed by purifying the eluted product via gel electrophoresis and cutting out the as well as premature termination, are common in synthetic DNA sequences. The
band with the correct length. Further characterization of synthetic oligonucleotides accumulation of errors from phosphoramidite chemical synthesis alone can lead to
may include sequencing or simply verifying the predicted molecular mass by mass only about 30% of any synthesized 100-mer being the desired sequence [33].
spectrometry. Improper annealing during oligo assembly can also introduce heterogeneity in the
final pool of synthetic gene products.

Cloning newly synthesized sequences into a plasmid vector can simplify the process
Step 3: Gene Assembly of sequence verification. Sequencing primers that bind to vector regions flanking the
gene insert ensure correct sequencing of the ends of the synthesized gene insert.
Many methods of assembling oligos into complete genes or larger genome building Further, plasmid DNA can be clonally amplified to create a homogeneous pool of
blocks have been developed and successfully used [16-32]. For relatively short DNA with the correct sequence.
sequences (up to 1 kb), polymerase-based or ligase-based in vitro assembly methods
are sufficient. For longer sequences, in vivo recombination-based methods may be In the event that the correct sequence cannot be obtained and amplified from the
preferred. Correct assembly requires a high-fidelity enzyme (e.g. DNA polymerase or pool of synthesized DNA, numerous methods for error detection and correction have
ligase). been developed and successfully used. These include stringent hybridization using
carefully designed oligos; exhaustive purification using electrophoresis, mass
Polymerase chain assembly (PCA) is a standard technique for polymerase-based spectrometry and other biochemical methods; mismatch-binding or
oligo assembly in a thermocycler. This reaction is also called templateless PCR. The mismatch-cleavage using prokaryotic endonucleases; selection of correct coding
principle is to combine all of the single-stranded oligos into a single tube, perform sequences via functional assays; and site-directed mutagenesis after sequencing
thermocycling to facilitate repeated rounds of annealing, extension, and [34]. PAGE or agarose gel purification is the technique in longest use, but it is costly
denaturation. Then use the outermost primers to amplify the full-length sequence. and labor intensive and does not identify or correct for substitutions or for small
The success of this method depends upon the accurate synthesis of oligos designed insertions/deletions. Enzyme-based strategies are limited by the enzyme’s
to possess sufficient regions of overlap, have sufficiently similar melting and capabilities; for example, the widely used E. coli MutHLS is not very effective for
annealing temperatures, and have minimal opportunities for mishybridization. substitutions other than G-T, A-C, G-G, A-A [34]. Site-directed mutagenesis after
Protocols for PCA to produce final sequences up to ~750 kb have been published[13]. sequencing can introduce point mutations using mutant primers and high-fidelity
DNA polymerase followed by selection for unmethylated molecules, but can be an
Sequence- and Ligation-Independent Cloning (SLIC) is a method of in vitro unwieldy technique. Because error correction can be so time-consuming and costly,
homologous recombination employing a T4 DNA polymerase that allows the especially for long or complex sequences, efforts continue to improve the accuracy
assembly of up to five gene fragments via simultaneous incorporation into a plasmid of oligo synthesis and assembly.

21 www.genscript.com www.genscript.com 22
GenEZTM Custom Molecular Clones and ORF Specify sequence &
Focus on other experiments while
GenScript
Receive sequence-verified
select vectors online plasmids in the mail
Clones Powered by Gene Synthesis does the cloning for you

What are Custom Clones? Table 2. Top advantages of GenEZTM Custom Molecular Clones
While it is possible to perform both traditional molecular cloning and gene synthesis
in the laboratory, both of these procedures can be tedious, time-consuming, and None. GenEZTM sequences are not restricted by
No sequence sequence complexity. If you can design it, we can
laborious. Fortunately a number of companies offer gene synthesis and custom restrictions synthesize it
cloning as a service, which delivers the desired gene in the desired vector in less time
than it would take to build the construct in the lab. Expansive vector 100 popular in-stock vectors or any vector of your
selection choice
Not only can cloning and gene synthesis services accelerate the generation of gene
constructs in the molecular biology lab, but they can allow for an influx of Sequence-verification Each clone is guaranteed 100% sequence-verified
contributions from scientists who may previously have been excluded from certain
interdisciplinary research due to their inexperience in molecular biology techniques. • Custom cloning
• Custom plasmid preparation (research and
For instance, a bioprocess engineer who conceives of a novel genetic circuit that can Downstream services transfection grade)
increase lipid production for a biofuel application may not necessarily need to • Mutagenesis service
become well-versed in molecular biology, nor purchase molecular biology research • Custom protein expression
equipment to implement his ideas. He could simply order the custom genetic circuits
that will function as he has designed, and then immediately begin his
experimentation.
What are ORF Clones?
Below we review two of the most powerful services in the custom cloning industry –
Despite the rise in popularity of fields such as genetic engineering and synthetic
GenEZTM Custom Molecular Clone and GenEZTM ORF Clone services. Powered by
biology, molecular cloning is still largely used for the expression of native proteins.
GenScript’s proprietary gene synthesis technologies, these services deliver
Structural and functional characterization of these proteins was spurred in part by
mutation-free gene sequences in a time and cost-effective manner.
the completion of the Human Genome Project, and more recently has been further
fueled by advances in proteomics and proteome mapping which postulate that the
GenEZTM Custom Molecular Clones human genome encodes for some 17,000+ proteins [35, 36].

– The next-generation of molecular cloning Native proteins and enzymes are encoded by open reading frame (ORF) sequences,
GenScript’s GenEZTM Custom Molecular Clone service combines GenScript’s which exclude introns, and 5' or 3' untranslated regions such as terminators and
proprietary gene synthesis technologies with custom cloning to deliver promoters. To facilitate protein expression, these protein encoding ORF sequences
sequence-verified, custom genes in your vector of choice in as few as 2 weeks. are cloned into expression vectors that may contain N- or C-terminal tags for protein
GenEZTM Molecular Clones are ideal if you desire non-natural engineered sequences, detection or purification.
mutant sequences, codon optimized sequences, or other customized recombinant
DNA constructs.

23 www.genscript.com www.genscript.com 24
GenEZTM ORF Clones – The easiest way to
Largest ORF database in the world
clone
• > 2 million ORFs from 186 species*
• > 40,000 ORFs in-stock
GenScript’s GenEZTM ORF Clone service is *Available for on-demand synthesis
powered by our proprietary gene synthesis
technology, which has allowed us to synthesize
over 40,000 ORF sequences de novo, which are
currently in stock. Our gene synthesis
technology has also allowed us to expand our
GenEZTM ORF Custom Cloning and Mutant
ORF clone collection to any protein encoding Clones
Cost-efficient
sequence registered in the NCBI RefSeq GenEZTM ORF clones are highly customizable and can be custom cloned into the
• Starting at $99/clone*
database – over 2 million sequences • Fully sequence verified vector of your choice, mutated, or undergo our downstream protein expression
synthesized on demand. GenEZTM ORF clones service.
can be shipped in as few as 5-days.

While most ORF clone databases are limited to human, mouse, or rat ORF
sequences, the combination of our in-stock clones and on demand sequence
synthesis capability affords our customers the opportunity to select their desired
ORF clone from the largest ORF sequence database in the world. GenEZTM ORF Clone

GenEZTM ORF species coverage

The GenEZTM ORF Clone database includes 2 million ORFs from 186 species including
but not limited to:

Human Mammals Plants

Mouse Ovis Aries Arabidopsis thaliana
Rat Oryctolagus cuniculus Chlamydomonas reinhardtii
Nicotiana benthamiana
Oryza sativa Subcloning Mutagenesis Protein expression

Vertebrates Invertebrates Fungi

Danio rerio Caenorhabditis elegans Saccharomyces cerevisiae Standard expression vector
Gallus gallus domesticus Drosophila melanogaster Schizosaccharomyces pombe GenScript’s GenEZTM ORF clones are seamlessly cloned into our standard mammalian
Xenopus laevis Neurospora crassa expression vector, pcDNA3.1+N-DYK using our CloneEZTM seamless cloning technology.
Driven by a CMV promoter, pcDNA3.1+N-DYK is equipped with an N-terminal
DYKDDDDK tag for easy protein detection and purification. GenEZTM ORF clones are
delivered expression and transfection ready, saving researchers valuable time in the
lab.

25 www.genscript.com www.genscript.com 26
 CMV p Nhe l
r rom pCDNA pUC pCold pENTR pGEX
p ote Afl ll
Am r Hind lll pET pBluescript pEGFP pFastBac PQE

pPIC pYES pGL pMAL

Ko
zak
in Start
pUC orig
In addition ORFs cloned into custom vectors can be further customized by the

DYK
addition of up to 30 bp of 5' or 3' flanking sequence – free of charge.
pcDNA3.1+N-DYK
Target ORF
Selected GenEZTM ORF Clone vectors
Stop
Bacterial
EcoR l Vector name Promoter Epitope tag Length
Selection
N Pst l
eo r pcDNA3.1+N-DYK CMV Ampicillin N-DYK 5453 bp
EcoR V
SV4 ..
0 pr o m oter . pcDNA3.1+N-HA CMV Ampicillin N-HA 5450 bp
pcDNA3.1+N-6His CMV Ampicillin N-6*HIS 5441 bp
pcDNA3.1+N-Myc CMV Ampicillin N-MYC 5453 bp
Vector name pcDNA3.1+-DYK pcDNA3.1+N-GST(a) CMV Ampicillin N-GST-(TEV) 6092 bp
Vector length (w/o insert) 5453 bp pcDNA3.1+N-GST(b) CMV Ampicillin N-GST-(Thrombin) 6086 bp
Promoter CMV pcDNA3.1+C-DYK CMV Ampicillin c-DYK 5438 bp

N-terminal flag DYDDDDK pcDNA3.1+C-HA CMV Ampicillin C-HA 5441 bp

pcDNA3.1+C-6His CMV Ampicillin C-6*HIS 5432 bp
Bacterial selection AmpR
pcDNA3.1+C-Myc CMV Ampicillin C-MYC 5444 bp
Mammalian selection NeoR

Mutant ORF Clones

Unlike ORF clones from other
Custom vectors companies, GenEZTM ORF clones can
For enhanced flexibility, GenEZTM ORF
undergo mutagenesis service to
clones can be directly custom cloned
create the mutant variants you need
into one of over 100 expression
for your functional studies.
vectors which are compatible with
mammalian, bacterial, and yeast Mutagenesis services include: Mutant ORFs
expression systems, including pcDNA, • Point mutations
pET, pPIC, and pUC vectors series.
Customized vectors • Mutant ORF clones from $149
• Deletions
• Only $50 for subcloning • Just 3 days additional production
• Insertions time
• Choose from > 100 expression
vectors • 5' or 3' Truncations
GenEZTM custom cloning vectors
• Add up to 30 bp of 5' and • Protein domains
include but are not limited to the 3' flanking sequence for free • 5' or 3' end sequence modifications
following vector series.

27 www.genscript.com www.genscript.com 28
GenEZTM ORF Clonesets References
Clonesets are large collections of related ORF clones that contain the key genes of 1. Meselson M. and Weigle J.J. (1961) Chromosome breakage accompanying genetic
biological signaling pathways or even whole genomes (also called ORFeomes). recombination in bacteriophage. Proc. Natl. Acad. Sci. USA 47, 857-868.
Clonesets are useful for high-throughput screening assays in the arenas of drug
2. Kellenberger G., Zichichi, M.L. and Weigle J.J. (1961) Exchange of DNA in the
discovery and systems biology. recombination of bacteriophage λ. Proc. Natl. Acad. Sci. USA 47, 869-878.

3. Campbell A. (1962) Episomes. Adv. Genet. 11, 101-145.

TM
Selected GenEZ Clonesets 4. Luria S.E. and Human M.L. (1952) A nonhereditary host-induced variation of
bacterial viruses. J. Bacteriol. 64, 557-569.
Pathway Species # of genes 5. Bertani G. and Weigle J.J. (1953) Host controlled variation in bacterial viruses. J.
Methylation Homo sapiens 10 Bacteriol. 65, 113-121.
ERK/MAPK targets Homo sapiens 21 6. Meselson M. and Yuan R. (1968) DNA restriction enzyme from E. coli. Nature. 217:
p38 MAPK signaling pathway Homo sapiens 34 1110−1114.
PI3K/AKT signalling Homo sapiens 102 7. Kelly T.J. and Smith HO. (1970) A restriction enzyme from Heomophilus influenzae.
mTOR signaling pathway Homo sapiens 61 II. Base sequence of the recognition site. Journal of Molecular Biology. 51: 393−409.
G1/S DNA damage checkpoints Homo sapiens 62 8. Cozzarelli N.R., Melechen N.E., Jovin T.M., and Kornberg A. (1967) Polynucleotide
G2/M DNA damage checkpoint Homo sapiens 9 cellulose as a substrate for a polynucleotide ligase induced by phage T4. Biochem.
Biophys. Res. Commun. 28, 578-586.
DNA damage response Homo sapiens 67
Signaling by GPCR Homo sapiens 1068 9. Gefter M.L., Becker A., and Hurwitz J. (1967) The enzymatic repair of DNA. I.
IL-6 Signaling Pathway Homo sapiens 99
Formation of circular lambda-DNA. Proc. Natl. Acad. Sci. USA 58, 240-247.

TNF-alpha/NF-kB signaling pathway Homo sapiens 196 10. Gellert M. (1967) Formation of covalent circles of lambda DNA by E. coli extracts.
TGF-beta signaling pathway Homo sapiens 80 Proc. Natl. Acad. Sci. USA 57, 148-155.
B cell receptor signaling pathway Homo sapiens 72 11. Olivera B.M. and Lehman I.R. (1967) Linkage of polynucleotides through
ErbB signaling pathway Homo sapiens 87 phosphodiester bonds by an enzyme from Escherichia coli. Proc. Natl. Acad. Sci. USA
57, 1426-1433.
Angiogenesis Homo sapiens 23
Wnt signaling pathway Homo sapiens 139 12. Weiss B. and Richardson C.C. (1967) Enzymatic breakage and joining of
deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system
Notch signaling pathway Homo sapiens 48 from Escherichia coli infected with T4 bacteriophage. Proc. Natl. Acad. Sci. USA 57,
Nuclear receptors Homo sapiens 38 1021-1028.

13. Burgess-Brown N.A. et al. ( 2008) Codon optimization can improve expression of
human genes in Escherichia coli: A multi-gene study. Protein Expr. Purif. 59 94-102.

14. Michelson, A.M. and Todd, A.R. Nucleotides part XXXII. Synthesis of a
dithymidine dinucleotide containing a 3: 5-internucleotidic linkage. Journal of the
Chemical Society (Resumed) 2632–2638 (1955).

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15. Agarwal, K.L. et al. Total synthesis of the gene for an alanine transfer ribonucleic interleukin-5. Protein Expr. Purif. 11, 86-94.
acid from yeast. Nature 227: 27–34 (1970).
30. Au L.C., Yang F.Y., Yang W.J., Lo S.H., and Kao C.F. (1998) Gene synthesis by a
16. Marchand J.A. and Peccoud J. (2012) Building block synthesis using the LCR-based approach: high-level production of leptin-L54 using synthetic gene in
polymerase chain assembly method. Methods Mol. Biol. 852, 3-10. Escherichia coli. Biochem. Biophys. Res. Commun. 248, 200-203.

17. Kelly J.R. et al. ( 2009) Measuring the activity of BioBrick promoters using an in 31. Li M.Z. and Elledge S.J. (2012) SLIC: a method for sequence- and
vivo reference standard. J. Biol. Eng. 3, 4. ligation-independent cloning. Methods Mol. Biol. 852, 51-59.

18. Gibson D.G. et al. (2008) Complete chemical synthesis, assembly, and cloning of 32. Ho-Shing O., Lau K.H., Vernon W., Eckdahl T.T. & Campbell A.M. (2012) Assembly
a Mycoplasma genitalium genome. Science 319, 1215-1220. of standardized DNA parts using BioBrick ends in E. coli. Methods Mol. Biol. 852,
61-76.
19. Horton R.M. et al. (1993) Gene splicing by overlap extension. , Gene splicing by
overlap extension. Meth. Enzymol. 217, 270-279. 33. Young L. and Dong Q. (2004) Two-step total gene synthesis method. Nucleic
Acids Res. 32, e59.
20. Stemmer W.P., Crameri A., Ha K.D., Brennan T.M. and Heyneker H.L. (1995)
Single-step assembly of a gene and entire plasmid from large numbers of 34. Hughes R.A., Miklos A.E., and Ellington A.D. (2011) Gene synthesis: methods and
oligodeoxyribonucleotides. Gene 164, 49-53. applications. Meth. Enzymol. 498, 277-309.

21. Wooddell C.I. and Burgess R.R. (1996) Use of asymmetric PCR to generate long 35. Ma S., Saaem I. and Tian J. (2012) Error correction in gene synthesis technology.
primers and single-stranded DNA for incorporating cross-linking analogs into specific Trends Biotechnol. 30, 147-154
sites in a DNA probe. Genome Res. 6, 886-892.
36. Wilhelm M. et al. (2014) Mass-spectrometry-based draft of the human
22. Gao X., Yo P., Keith A., Ragan T.J., and Harris T.K. (2003) Thermodynamically proteome. Nature 509, 582-587.
balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer
design for high-fidelity assembly of longer gene sequences. Nucleic Acids Res. 31, 37. Kim M-S. (2014) A draft map of the human proteome. Nature 509, 575–581.
e143.

23. Tian J. et al. (2004) Accurate multiplex gene synthesis from programmable DNA
microchips. Nature 432, 1050-1054.

24. Wu G. et al. (2006) Simplified gene synthesis: a one-step approach to PCR-based

gene construction. J. Biotechnol. 124, 496-503.

25. Kong D.S., Carr P.A., Chen L., Zhang S. & Jacobson J.M. (2007) Parallel gene
synthesis in a microfluidic device. Nucleic Acids Res. 35, e61.

26. Yehezkel T.B., Linshiz G., Buaron H., Kaplan S., Shabi U., and Shapiro E. (2008) De
novo DNA synthesis using single molecule PCR. Nucleic Acids Res. 36, e107.

27. Huang M.C., Cheong, W.C., Ye, H. & Li M.-H. (2012) Top down real-time gene
synthesis. Methods Mol. Biol. 852, 23-34.

28. Eren M. & Swenson R. P. ( 1989) Chemicalsynthesis and expression of a synthetic

gene for the flavodoxin from Clostridium MP. J. Biol. Chem. 264, 14874-14879.

29. Mehta D.V., DiGate R.J., Banville D.L. & Guiles R.D. ( 1997) Optimized gene
synthesis, high level expression, isotopic enrichment, and refolding of human

31 www.genscript.com www.genscript.com 32