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
Difference in DNA polymerase behaviors.The tetracycline resistance gene assembly product was PCR amplified by Taq DNA polymerase (Lane 1) and Pfx DNA polymerase (Lane 2). Pfx amplification caused polymerization and produced some high molecular weight products, thus the Taq polymerase was chosen for subsequent studies. The last lane contained the 100-bp DNA ladder.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145682.g004: Difference in DNA polymerase behaviors.The tetracycline resistance gene assembly product was PCR amplified by Taq DNA polymerase (Lane 1) and Pfx DNA polymerase (Lane 2). Pfx amplification caused polymerization and produced some high molecular weight products, thus the Taq polymerase was chosen for subsequent studies. The last lane contained the 100-bp DNA ladder.

Mentions: We also tried to discern if there are any assembly error patterns. Most of the errors in the assembled genes were single-base mutations or deletions. Originally, we thought some errors could occur in the fragment junctions because these are where the assembly activities happened. However, we found errors can occur anywhere in the fragments, including those interior regions protected by double-strand. Some of these errors could have been present in the original oligonucleotides that were used for assembly because it is known that oligonucleotide synthesis can be imperfect [29]. We also suspect that some of the errors might have been introduced during the PCR amplification of assembled products. We have used both the Taq DNA polymerase (NEB #M0273) and Pfx high fidelity DNA polymerase (Life Technologies #11708) for the PCRs, and we have found Pfx can produce polymerization products with high molecular weight (Fig 4), so we mainly used Taq in most of our studies. Since Taq has no 3’ exonuclease proof-reading activity, that may have increased the errors due to lingering unassembled fragments in the buffer. If the PCR amplification step can be avoided, it is reasonable to expect that fewer assembly errors will occur. We are going to study other assembly methods that might allow higher fragment concentration to begin with, thus the assembled products may not require PCR amplification. If successful, this will improve the assembly quality.


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)

Difference in DNA polymerase behaviors.The tetracycline resistance gene assembly product was PCR amplified by Taq DNA polymerase (Lane 1) and Pfx DNA polymerase (Lane 2). Pfx amplification caused polymerization and produced some high molecular weight products, thus the Taq polymerase was chosen for subsequent studies. The last lane contained the 100-bp DNA ladder.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145682.g004: Difference in DNA polymerase behaviors.The tetracycline resistance gene assembly product was PCR amplified by Taq DNA polymerase (Lane 1) and Pfx DNA polymerase (Lane 2). Pfx amplification caused polymerization and produced some high molecular weight products, thus the Taq polymerase was chosen for subsequent studies. The last lane contained the 100-bp DNA ladder.
Mentions: We also tried to discern if there are any assembly error patterns. Most of the errors in the assembled genes were single-base mutations or deletions. Originally, we thought some errors could occur in the fragment junctions because these are where the assembly activities happened. However, we found errors can occur anywhere in the fragments, including those interior regions protected by double-strand. Some of these errors could have been present in the original oligonucleotides that were used for assembly because it is known that oligonucleotide synthesis can be imperfect [29]. We also suspect that some of the errors might have been introduced during the PCR amplification of assembled products. We have used both the Taq DNA polymerase (NEB #M0273) and Pfx high fidelity DNA polymerase (Life Technologies #11708) for the PCRs, and we have found Pfx can produce polymerization products with high molecular weight (Fig 4), so we mainly used Taq in most of our studies. Since Taq has no 3’ exonuclease proof-reading activity, that may have increased the errors due to lingering unassembled fragments in the buffer. If the PCR amplification step can be avoided, it is reasonable to expect that fewer assembly errors will occur. We are going to study other assembly methods that might allow higher fragment concentration to begin with, thus the assembled products may not require PCR amplification. If successful, this will improve the assembly quality.

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