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Background: 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. Results: We 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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]