Abstract
BackgroundInsertion of engineered DNA fragments into bacterial vectors is the foundation of recombinant DNA technology, yet existing methods are still laborious, require many steps, depend on specific vector configuration, or require expensive reagents.ResultsWe have developed a method, called “Pyrite” cloning that combines the traditional restriction enzyme digestion and ligation reaction in a single tube and uses a programmed thermocycler reaction, allowing rapid and flexible cloning in a single tube. After the Pyrite reaction and transformation, approximately 50% colonies contain the expected insert, which can be easily and quickly determined by colony PCR or blue-white colony screening. We also demonstrated that Pyrite cloning can be applied for different cloning purposes.ConclusionsThe Pyrite cloning method reported here is a single tube and programmed reaction cloning with restriction enzymes. Compared to other cloning methods, Pyrite cloning is flexible, inexpensive, simple, and highly efficient.
Highlights
Insertion of engineered DNA fragments into bacterial vectors is the foundation of recombinant DNA technology, yet existing methods are still laborious, require many steps, depend on specific vector configuration, or require expensive reagents
Though traditional restriction enzyme digestion and ligation has long served as the original recombinant DNA method, other cloning methods have been developed to date, such as the Gibson assembly [4], Gateway system [6], and Golden Gate cloning [3]
The same limitation exists for Advanced QUick Assembly (AQUA) cloning, Ligation-Independent Cloning (LIC), and Sequence- and Ligation-Independent Cloning (SLIC), as all three methods depend on overlaps that often restrict destination vector selection to one option [1, 2, 5]
Summary
To get more accurate Pyrite cloning efficiency, the blue/white colony screen method was used with the vector pUC19, which contains a lacZ gene that is disrupted by insertion [8]. A single Pyrite cloning reaction may be utilized to insert a fragment simultaneously into two different vectors containing different antibiotic resistance genes (Fig. 3b). To test blue/white screening, F. vesca gene31413 CDS was cloned into two vectors simultaneously, pUC19 (Fig. 2f; Table 1) and pSanFran (Table 1), and both yielded 100% white colonies. We used the blue/white screen to test the efficiency of swapping eGFP from the pSanFran vector to pUC19 in a 1:1 pmol ratio with the Pyrite reaction, which yielded 48.3% white colonies (Fig. 2g; Table 1).
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