Limits...
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.

Show MeSH
Assembly efficiency and reaction count under different conditions.(a) The assembly efficiency and fragment count to assemble a 2000 bp gene using different fragment sizes. (b) The assembly reactions required to assemble sequences up to a million bps from 200 bp fragments under different Gibson Assembly capacities up to 30 fragments at once. In both figures the junction length between fragments is fixed at 20 bps and it is assumed that any sequence to assemble can be evenly divided by the fragments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145682.g003: Assembly efficiency and reaction count under different conditions.(a) The assembly efficiency and fragment count to assemble a 2000 bp gene using different fragment sizes. (b) The assembly reactions required to assemble sequences up to a million bps from 200 bp fragments under different Gibson Assembly capacities up to 30 fragments at once. In both figures the junction length between fragments is fixed at 20 bps and it is assumed that any sequence to assemble can be evenly divided by the fragments.

Mentions: In this study, our main focus is to test if we can significantly increase the number of dsDNA fragments that can be assembled at once. The reason we chose to create shorter dsDNA fragments from complementary oligonucleotides is to avoid increasing the length of the assembled gene products—it will cost a lot more to synthesize longer dsDNA fragments and the assembled sequences will be harder to validate if they must go through shotgun assembly. We must point out that this is our strategy to test high-number assemblies and is not a very efficient way to directly assemble genes because a significant fraction of junction regions were synthesized twice and wasted. In practice, the longer the individual fragments, the more efficient the assembled sequences can be elongated. Assuming 20 bp junctions between all fragments and a 2000 bp gene can be evenly divided among fragments of any sizes, as seen in Fig 3, it requires 80 fragments of 45 bps to assemble the gene with an efficiency less than 56%, but it only requires 12 fragments of 200 bps to assemble the same gene with an efficiency above 90%. Because the interior of each DNA fragment is protected by double-strand and does not interfere with the assembly process, in principle the length of each fragment should not significantly increase the assembly difficulty. Therefore, longer genes can be assembled using longer fragments when designed with our method. Further studies will have to be conducted to test high-number gene assemblies using longer fragments.


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)

Assembly efficiency and reaction count under different conditions.(a) The assembly efficiency and fragment count to assemble a 2000 bp gene using different fragment sizes. (b) The assembly reactions required to assemble sequences up to a million bps from 200 bp fragments under different Gibson Assembly capacities up to 30 fragments at once. In both figures the junction length between fragments is fixed at 20 bps and it is assumed that any sequence to assemble can be evenly divided by the fragments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696799&req=5

pone.0145682.g003: Assembly efficiency and reaction count under different conditions.(a) The assembly efficiency and fragment count to assemble a 2000 bp gene using different fragment sizes. (b) The assembly reactions required to assemble sequences up to a million bps from 200 bp fragments under different Gibson Assembly capacities up to 30 fragments at once. In both figures the junction length between fragments is fixed at 20 bps and it is assumed that any sequence to assemble can be evenly divided by the fragments.
Mentions: In this study, our main focus is to test if we can significantly increase the number of dsDNA fragments that can be assembled at once. The reason we chose to create shorter dsDNA fragments from complementary oligonucleotides is to avoid increasing the length of the assembled gene products—it will cost a lot more to synthesize longer dsDNA fragments and the assembled sequences will be harder to validate if they must go through shotgun assembly. We must point out that this is our strategy to test high-number assemblies and is not a very efficient way to directly assemble genes because a significant fraction of junction regions were synthesized twice and wasted. In practice, the longer the individual fragments, the more efficient the assembled sequences can be elongated. Assuming 20 bp junctions between all fragments and a 2000 bp gene can be evenly divided among fragments of any sizes, as seen in Fig 3, it requires 80 fragments of 45 bps to assemble the gene with an efficiency less than 56%, but it only requires 12 fragments of 200 bps to assemble the same gene with an efficiency above 90%. Because the interior of each DNA fragment is protected by double-strand and does not interfere with the assembly process, in principle the length of each fragment should not significantly increase the assembly difficulty. Therefore, longer genes can be assembled using longer fragments when designed with our method. Further studies will have to be conducted to test high-number gene assemblies using longer fragments.

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