ArticleBiological procedures online2013
Simultaneous splicing of multiple DNA fragments in one PCR reaction.
Article in Biological procedures online, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
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- Reprogramming the EnvZ-OmpR two-component system confers ethanol tolerance in Escherichia coli by stabilizing the outer membrane and altering ferric homeostasis.PLoS genetics · 2025Article
- Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against lethal Acinetobacter baumannii infection.Scientific reports · 2024Article
- A Collagen10a1 mutation disrupts cell polarity in a medaka model for metaphyseal chondrodysplasia type Schmid.iScience · 2024Article
- A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death.Journal of translational medicine · 2022Article
- Incorporation of a truncated form of flagellin (TFlg) into porcine circovirus type 2 virus-like particles enhances immune responses in mice.BMC veterinary research · 2020Article
- Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.Human gene therapy · 2015Review
- A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments.Avicenna journal of medical biotechnologyArticle
Corrections and comments
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundRapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments.
resultsWe performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5-10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification.
conclusionsOur method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match.
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Registered trials
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