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Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

Birla BS, Chou HH - PLoS ONE (2015)

Bottom Line: Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning.Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor.In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa, United States of America.

ABSTRACT
Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

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The Gibson Assembly method and Picky thermodynamic junction analysis.(a) The Gibson Assembly reagent includes three enzymes. The 5’ exonuclease erodes the 5’ ends on each dsDNA fragment, exposing single-stranded 3’ overhangs. The overhangs anneal to each other according to their compatible base-pairing. The DNA polymerase repairs gaps and the DNA ligase covalently binds the fragments to create a full-length product. (b) To design an optimal fragment set for gene assembly, the target gene is first analyzed using the Picky software to identify all its thermodynamically unique junction regions. Next, a separate Perl program takes these junction coordinates as well as some user specified design parameters such as acceptable minimum and maximum fragment lengths and the optimization goal for lower cost or fewer fragment count to finalize the optimal fragment set.
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pone.0145682.g001: The Gibson Assembly method and Picky thermodynamic junction analysis.(a) The Gibson Assembly reagent includes three enzymes. The 5’ exonuclease erodes the 5’ ends on each dsDNA fragment, exposing single-stranded 3’ overhangs. The overhangs anneal to each other according to their compatible base-pairing. The DNA polymerase repairs gaps and the DNA ligase covalently binds the fragments to create a full-length product. (b) To design an optimal fragment set for gene assembly, the target gene is first analyzed using the Picky software to identify all its thermodynamically unique junction regions. Next, a separate Perl program takes these junction coordinates as well as some user specified design parameters such as acceptable minimum and maximum fragment lengths and the optimization goal for lower cost or fewer fragment count to finalize the optimal fragment set.

Mentions: Synthetic biology is a new research field involving genes and genomes that are artificially designed, constructed and transformed into living cells [1]. The direct synthesis approach is preferable even for naturally occurring genes that can be cloned using recombinant DNA technologies, because it is often more efficient, reliable and flexible to synthesize genes rather than to clone them. Synthetic biology requires multiple hierarchical levels of assemblies starting from the smallest building blocks of short oligonucleotides to eventually reaching a full-length genome [2]. Several different gene assembly methods have been developed, including ligation-dependent assembly methods [3–6], the FokI method [7], variations of PCR-based methods [8–15], the BioBrickTM assembly method [16,17], in vivo recombinant assembly methods [18,19] and the ligation cycling reaction method [20]. Among gene assembly methods, the relatively recent Gibson Assembly is one of the easiest ones to use [21,22], and has become a commercially available reagent kit from New England Biolabs (NEB Gibson Assembly Master Mix, #E2011). In the Gibson Assembly (see Fig 1A), three different DNA enzymes are optimally mixed together to assemble double-stranded (ds) DNA fragments: 1) a 5’ exonuclease, which shortens the 5’ end of DNA fragments and exposes a single-stranded 3’ overhang that can anneal to the other exposed DNA strands; 2) a DNA polymerase that fills in the missing DNA nucleotides after two strand annealing to repair the gaps; and 3) a DNA ligase that covalently repairs the nicks between two adjacent DNA fragments to make a single DNA molecule. Gibson Assembly has the following benefits: 1) the interior part of each DNA fragment is protected and cannot cause incorrect assembly because it remains double-stranded throughout the assembly process. This is in stark contrast to PCR based assembly methods where all strands in all DNA fragments are accessible to unintended hybridizations and may cause mis-assemblies during the repeated denaturing and re-annealing cycles; 2) Because all enzymes and DNA fragments required for assembly are mixed in at once, Gibson Assembly requires just a single step, a single tube, and about an hour reaction time; 3) It does not depend on specific DNA sequences (e.g., restriction enzyme recognition sites) for the assembly and it does not produce any scar in the resulted sequence; and 4) The assembled product can be used directly for many downstream steps, e.g., bacteria transformation (if a vector backbone is included in the DNA fragments assembled), restriction digestion for cloning, and PCR amplification.


Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

Birla BS, Chou HH - PLoS ONE (2015)

The Gibson Assembly method and Picky thermodynamic junction analysis.(a) The Gibson Assembly reagent includes three enzymes. The 5’ exonuclease erodes the 5’ ends on each dsDNA fragment, exposing single-stranded 3’ overhangs. The overhangs anneal to each other according to their compatible base-pairing. The DNA polymerase repairs gaps and the DNA ligase covalently binds the fragments to create a full-length product. (b) To design an optimal fragment set for gene assembly, the target gene is first analyzed using the Picky software to identify all its thermodynamically unique junction regions. Next, a separate Perl program takes these junction coordinates as well as some user specified design parameters such as acceptable minimum and maximum fragment lengths and the optimization goal for lower cost or fewer fragment count to finalize the optimal fragment set.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4696799&req=5

pone.0145682.g001: The Gibson Assembly method and Picky thermodynamic junction analysis.(a) The Gibson Assembly reagent includes three enzymes. The 5’ exonuclease erodes the 5’ ends on each dsDNA fragment, exposing single-stranded 3’ overhangs. The overhangs anneal to each other according to their compatible base-pairing. The DNA polymerase repairs gaps and the DNA ligase covalently binds the fragments to create a full-length product. (b) To design an optimal fragment set for gene assembly, the target gene is first analyzed using the Picky software to identify all its thermodynamically unique junction regions. Next, a separate Perl program takes these junction coordinates as well as some user specified design parameters such as acceptable minimum and maximum fragment lengths and the optimization goal for lower cost or fewer fragment count to finalize the optimal fragment set.
Mentions: Synthetic biology is a new research field involving genes and genomes that are artificially designed, constructed and transformed into living cells [1]. The direct synthesis approach is preferable even for naturally occurring genes that can be cloned using recombinant DNA technologies, because it is often more efficient, reliable and flexible to synthesize genes rather than to clone them. Synthetic biology requires multiple hierarchical levels of assemblies starting from the smallest building blocks of short oligonucleotides to eventually reaching a full-length genome [2]. Several different gene assembly methods have been developed, including ligation-dependent assembly methods [3–6], the FokI method [7], variations of PCR-based methods [8–15], the BioBrickTM assembly method [16,17], in vivo recombinant assembly methods [18,19] and the ligation cycling reaction method [20]. Among gene assembly methods, the relatively recent Gibson Assembly is one of the easiest ones to use [21,22], and has become a commercially available reagent kit from New England Biolabs (NEB Gibson Assembly Master Mix, #E2011). In the Gibson Assembly (see Fig 1A), three different DNA enzymes are optimally mixed together to assemble double-stranded (ds) DNA fragments: 1) a 5’ exonuclease, which shortens the 5’ end of DNA fragments and exposes a single-stranded 3’ overhang that can anneal to the other exposed DNA strands; 2) a DNA polymerase that fills in the missing DNA nucleotides after two strand annealing to repair the gaps; and 3) a DNA ligase that covalently repairs the nicks between two adjacent DNA fragments to make a single DNA molecule. Gibson Assembly has the following benefits: 1) the interior part of each DNA fragment is protected and cannot cause incorrect assembly because it remains double-stranded throughout the assembly process. This is in stark contrast to PCR based assembly methods where all strands in all DNA fragments are accessible to unintended hybridizations and may cause mis-assemblies during the repeated denaturing and re-annealing cycles; 2) Because all enzymes and DNA fragments required for assembly are mixed in at once, Gibson Assembly requires just a single step, a single tube, and about an hour reaction time; 3) It does not depend on specific DNA sequences (e.g., restriction enzyme recognition sites) for the assembly and it does not produce any scar in the resulted sequence; and 4) The assembled product can be used directly for many downstream steps, e.g., bacteria transformation (if a vector backbone is included in the DNA fragments assembled), restriction digestion for cloning, and PCR amplification.

Bottom Line: Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning.Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor.In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa, United States of America.

ABSTRACT
Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

Show MeSH