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  • ‹ Back to In-Fusion Cloning and competition
  • In-Fusion Snap Assembly vs. GeneArt Gibson Assembly HiFi
  • In-Fusion Snap Assembly vs. NEBuilder HiFi
  • Sequence accuracy in seamless cloning
  • Choosing a seamless cloning method
  • Improving background over Gibson Assembly
  • A successful alternative to ligation cloning
  • Single- and multiple-insert cloning
  • Easy cloning into lentiviral vectors
  • Outperforming TOPO cloning
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In-Fusion Cloning citations
In-Fusion Cloning Product info: In-Fusion Snap Assembly kits
In-Fusion Cloning Interest Sign up for In-Fusion updates and promotions
Home › Learning centers › Cloning › In-Fusion Cloning general information › In-Fusion Cloning and competition › Sequence accuracy in seamless cloning

In-Fusion Cloning general information

  • In-Fusion Cloning overview
  • In-Fusion Cloning guide
  • In-Fusion Cloning and competition
    • In-Fusion Snap Assembly vs. GeneArt Gibson Assembly HiFi
    • In-Fusion Snap Assembly vs. NEBuilder HiFi
    • Sequence accuracy in seamless cloning
    • Choosing a seamless cloning method
    • Improving background over Gibson Assembly
    • A successful alternative to ligation cloning
    • Single- and multiple-insert cloning
    • Easy cloning into lentiviral vectors
    • Outperforming TOPO cloning
  • In-Fusion Cloning citations
  • Stellar Competent Cells product overview and performance data
  • EcoDry reagents and sustainability
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Citations
In-Fusion Cloning citations
In-Fusion Cloning Product info: In-Fusion Snap Assembly kits
In-Fusion Cloning Interest Sign up for In-Fusion updates and promotions
Tech Note

Higher sequence accuracy for seamless constructs with In-Fusion Cloning

  • Seamless cloning enables directional, multiple-insert cloning without scar sequences
  • Methods for seamless cloning vary in their enzymatic mechanisms
  • Sequence accuracy is improved with ligase-free In-Fusion Cloning compared to Gibson Assembly and NEBuilder kits

Introduction Results Conclusions Methods References

Introduction  

Researchers have several options when it comes to seamless cloning methods. The mechanism for each varies quite a bit, as does the resulting sequence accuracy, which we will discuss below. The benefits to any seamless method include the lack of unwanted nucleotides—or scar sequences—between vector and insert, as well as the ability to clone multiple inserts in a specific order in just one round of cloning. However, these research tools lose some of their power if accuracy and/or efficiency suffer. High-throughput applications are particularly affected by any pain points in this regard, as problems with potential error rates or background levels will be magnified when projects are scaled up to accommodate larger cloning needs.

Several commercially available seamless cloning kits, including Gibson Assembly and NEBuilder, utilize a three-enzyme mix consisting of an exonuclease, polymerase, and ligase (Gibson et al. 2009). The mechanism for In-Fusion Cloning (Irwin et al. 2012) differs from this process in a simple but significant way. Both mechanisms require that inserts are amplified by PCR such that they include homologous overlaps with a linear vector or other adjacent fragments, and both utilize an exonuclease to chew back the DNA of linear fragments to create cohesive sticky ends. At this point, the workflows part ways. With the three-enzyme methods, a polymerase fills in any nucleotide gaps, and a DNA ligase then seals the nicks in the annealed fragments prior to the transformation step. This process runs the risk of introducing sequence errors and mismatches at the cloning junction. In contrast, In-Fusion technology does not use a polymerase nor DNA ligase in the cloning mechanism, thus eliminating the potential for nucleotide errors. Cohesive bonds are formed in vivo, preserving the sequence integrity of the clone. In an effort to understand the repercussions of the differences in these mechanisms, we used commercially available kits for each to perform the demanding task of multiple-insert cloning, specifically looking at sequence accuracy as a measure of success.

Results  

In the experiment below, we compared two kits for the three-enzyme method (NEBuilder DNA HiFi Assembly from NEB and the Gibson Assembly Kit from SGI) with the liquid and lyophilized (EcoDry) versions of Takara Bio's In-Fusion Cloning HD Plus kits in order to assess the sequence accuracy of clones made with each method. Each seamless cloning kit was used to clone five inserts (947 bp, 717 bp, 697 bp, 405 bp, and 1,005 bp) into a 2.7-kb vector backbone, with the molar ratio of vector to insert set at 1:2 (Table I).

Table I. Cloning reaction setup to achieve a vector-to-insert molar ratio of 1:2
Linear fragment Amount
Insert 1 (947 bp) 40 ng
Insert 2 (717 bp) 40 ng
Insert 3 (697 bp) 32 ng
Insert 4 (405 bp) 20 ng
Insert 5 (1,005 bp) 45 ng
Vector (2,671 bp) 37 ng

All clones were designed with 20-bp homologous overlaps between each pair of linear fragments, as per the recommendations for multiple-insert reactions with In-Fusion Cloning. Reactions for each system were carried out per their respective manufacturer's instructions, and 1/10 dilutions were plated on selective medium. 20 clones from each kit were chosen at random in order to check sequence fidelity. Both three-enzyme methods yielded clones with an average of 80% accuracy, while In-Fusion Cloning produced a higher level of accuracy (Table II), with liquid and EcoDry kits yielding 90% and 100%, respectively.

Table II. Sequence accuracy comparison for all four seamless cloning kits
Commercial kit Sequence accuracy (average of 20 clones)
In-Fusion HD Cloning Plus (liquid format) 90%
In-Fusion HD EcoDry Cloning Plus (lyophilized format) 100%
NEBuilder HiFi DNA Assembly Cloning Kit (NEB) 80%
Gibson Assembly Kit (SGI) 80%

Conclusions  

The sequence accuracy of In-Fusion Cloning is consistently higher than both commercial kits for three-enzyme cloning methods, regardless of which format of the In-Fusion enzyme mix is used. It is likely that this difference stems from the lack of ligase and polymerase in the In-Fusion Cloning mix, as both ligase and polymerase performance can be error-prone. Sequence errors can be especially problematic in multiple-insert cloning, as there are more cloning junctions where nucleotide mismatches can occur. The reliable results provided by In-Fusion technology make it especially adaptable to high-throughput workflows. Higher accuracy means less screening and fewer missed clones in large batches, which would then require additional rounds of cloning to recover.

Methods  

Each seamless cloning kit was used according to the manufacturer's instructions. Vector-to-insert molar ratios were set at 1:2 for each reaction. Reactions for each kit were run in triplicate, with the exception of the liquid In-Fusion Cloning kit, which was performed once to reproduce previous data. NEB 5-alpha Competent E. coli (High Efficiency; NEB) were transformed with the cloning reactions from the NEBuilder HiFi DNA Assembly Cloning Kit (NEB), TransforMax EPI300 Electrocompetent and Chemically Competent E. coli were transformed with the cloning reactions from the Gibson Assembly Kit (SGI), and Stellar Competent Cells (included in the In-Fusion Cloning kits) were transformed with the cloning reactions from both In-Fusion HD Cloning Plus and In-Fusion HD EcoDry Cloning Plus. 1/10 dilutions were plated on selective medium for each transformation. 20 clones were chosen at random from transformation plates of each cloning kit and checked for sequence accuracy via Sanger sequencing.

References  

Gibson D.G., et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6(5): 343–345 (2009).

Irwin C.R., Farmer A., Willer D.O., Evans D.H. In-Fusion Cloning with Vaccinia Virus DNA Polymerase. Vaccinia Virus and Poxvirology (Methods and Protocols). 890:23–35 (2012).

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License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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638948: In-Fusion Snap Assembly Master Mix

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The In-Fusion cloning protocol

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

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638953: In-Fusion Snap Assembly EcoDry Master Mix with Competent Cells

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The In-Fusion cloning protocol

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638954: In-Fusion Snap Assembly EcoDry Master Mix

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

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In-Fusion Cloning for high-throughput antibody development

A rapid, high-throughput cloning workflow for antibody development

Generating monoclonal antibodies requires efficient and rapid protocols for cloning, expression, and screening. Traditional cloning creates bottlenecks in this research, as it is often hampered by lengthy protocols, low efficiency, and reading frame errors. See how one lab has streamlined the process with In-Fusion technology, a highly accurate, seamless cloning method which is easily adaptable to high-throughput workflows.

Learn more

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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  • Products
  • COVID-19 research
  • Next-generation sequencing
  • Diagnostic solutions
  • Real-time PCR
  • Stem cell research
  • mRNA and cDNA synthesis
  • PCR
  • Cloning
  • Nucleic acid purification
  • Gene function
  • Protein research
  • Antibodies and ELISA
  • New products
  • Special offers
  • COVID-19 research
  • Viral detection with qPCR
  • SARS-CoV-2 pseudovirus
  • Human ACE2 stable cell line
  • Viral RNA isolation
  • Viral and host sequencing
  • Vaccine development
  • CRISPR screening
  • Drug discovery
  • Immune profiling
  • Publications
  • Next-generation sequencing
  • RNA-seq
  • DNA-seq
  • Single-cell NGS automation
  • Reproductive health
  • Bioinformatics tools
  • Whole genome amplification
  • Immune profiling
  • Diagnostic solutions
  • Reproductive health
  • Real-time PCR
  • Real-time PCR kits
  • Reverse transcription prior to qPCR
  • High-throughput qPCR solutions
  • RNA extraction and analysis for real-time qPCR
  • Stem cell research
  • Media and supplements
  • Stem cells and stem cell-derived cells
  • Single-cell cloning of edited hiPS cells
  • mRNA and cDNA synthesis
  • In vitro transcription
  • cDNA synthesis kits
  • Reverse transcriptases
  • RACE kits
  • Purified cDNA & genomic DNA
  • Purified total RNA and mRNA
  • PCR
  • Most popular polymerases
  • High-yield PCR
  • High-fidelity PCR
  • GC rich PCR
  • PCR master mixes
  • Cloning
  • In-Fusion seamless cloning
  • Competent cells
  • Ligation kits
  • Restriction enzymes
  • Nucleic acid purification
  • Plasmid purification kits
  • Genomic DNA purification kits
  • DNA cleanup kits
  • RNA purification kits
  • Cell-free DNA purification kits
  • Microbiome
  • Gene function
  • Gene editing
  • Viral transduction
  • Fluorescent proteins
  • T-cell transduction and culture
  • Tet-inducible expression systems
  • Transfection reagents
  • Cell biology assays
  • Protein research
  • Purification products
  • Two-hybrid and one-hybrid systems
  • Mass spectrometry reagents
  • Antibodies and ELISA
  • Primary antibodies and ELISAs by research area
  • Fluorescent protein antibodies
  • Special offers
  • Free samples
  • TB Green qPCR sale
  • PrimeSTAR enzyme promo
  • Try BcaBEST DNA Polymerase ver.2.0
  • RNA purification sale
  • Capturem IP and Co-IP sale
  • Baculovirus titration kits early access program
  • NGS bundle and save
  • Free sample: PrimePath Direct Saliva SARS-CoV-2 Detection Kit
  • TALON his-tag purification resin special offer
  • GoStix Plus special offers
  • PCR samples
  • Services & Support
  • Instrument services
  • Stem cell services
  • Gene and cell therapy manufacturing
  • Customer service
  • Sales
  • Shipping & delivery
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  • Feedback
  • Online tools
  • Partnering & Licensing
  • Vector information
  • Instrument services
  • Apollo services
  • ICELL8 services
  • SmartChip services
  • Stem cell services
  • Clinical-grade stem cell services
  • Research-grade stem cell services
  • Outsourcing stem cell-based disease model development
  • Gene and cell therapy manufacturing
  • Services
  • Facilities
  • Our process
  • Resources
  • Sales
  • Make an appointment with your sales rep
  • Online tools
  • GoStix Plus FAQs
  • Vector information
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  • Vector document finder
  • Learning centers
  • Automation systems
  • Next-generation sequencing
  • cDNA synthesis
  • Real-time PCR
  • Nucleic acid purification
  • PCR
  • Cloning
  • Stem cell research
  • Gene function
  • Protein research
  • Antibodies and ELISA
  • Automation systems
  • SmartChip Real-Time PCR System introduction
  • ICELL8 introduction
  • Next-generation sequencing
  • Technical notes
  • Featured kits
  • Technology and application overviews
  • FAQs and tips
  • DNA-seq protocols
  • Bioinformatics resources
  • Webinars
  • Real-time PCR
  • Overview
  • Reaction size guidelines
  • Guest webinar: extraction-free SARS-CoV-2 detection
  • Guest webinar: developing and validating molecular diagnostic tests
  • Technical notes
  • Nucleic acid purification
  • Nucleic acid extraction webinars
  • Product demonstration videos
  • Product finder
  • Plasmid kit selection guide
  • RNA purification kit finder
  • PCR
  • Citations
  • Selection guides
  • Technical notes
  • FAQ
  • Cloning
  • In-Fusion Cloning general information
  • Primer design and other tools
  • In‑Fusion Cloning tips and FAQs
  • Applications and technical notes
  • Stem cell research
  • Overview
  • Protocols
  • Technical notes
  • Gene function
  • Gene editing
  • Viral transduction
  • T-cell transduction and culture
  • Inducible systems
  • Cell biology assays
  • Protein research
  • Capturem technology
  • Antibody immunoprecipitation
  • His-tag purification
  • Other tag purification
  • Expression systems
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  • Interview: adapting to change with Takara Bio
  • Applications
  • Solutions
  • Partnering
  • Webinar: Speeding up diagnostic development
  • Contact us
  • Vaccine development
  • Characterizing the viral genome and host response
  • Identifying and cloning vaccine targets
  • Expressing and purifying vaccine targets
  • Immunizing mice and optimizing vaccine targets
  • Pathogen detection
  • Sample prep
  • Detection methods
  • Identification and characterization
  • SARS-CoV-2
  • Antibiotic-resistant bacteria
  • Food crop pathogens
  • Waterborne disease outbreaks
  • Viral-induced cancer
  • Immunotherapy research
  • T-cell therapy
  • Antibody therapeutics
  • T-cell receptor profiling
  • TBI initiatives in cancer therapy
  • Cancer research
  • Sample prep from FFPE tissue
  • Sample prep from plasma
  • Cancer biomarker discovery
  • Cancer biomarker quantification
  • Single cancer cell analysis
  • Cancer genomics and epigenomics
  • HLA typing in cancer
  • Gene editing for cancer therapy/drug discovery
  • Alzheimer's disease research
  • Antibody engineering
  • Sample prep from FFPE tissue
  • Single-cell sequencing
  • Reproductive health technologies
  • Preimplantation genetic testing
  • ESM Collection Kit forms
  • About
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  • That's Good Science!
  • Our brands
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  • Careers
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  • Quality and compliance
  • Takara Bio affiliates & distributors
  • Need help?
  • Website FAQs
  • BioView blog
  • Automation
  • Cancer research
  • Career spotlights
  • Current events
  • Customer stories
  • Gene editing
  • Research news
  • Single-cell analysis
  • Stem cell research
  • Tips and troubleshooting
  • Women in STEM
  • That's Good Support!
  • About our blog
  • That's Good Science!
  • SMART-Seq Pro Biomarker Discovery Contest
  • DNA extraction educational activity
  • That's Good Science Podcast
  • Season one
  • Season two
  • Season three
  • Our brands
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