[Frontiers in Bioscience E2, 1105-1114, June 1, 2010]

SMC, a simple method to rapidly assemble multiple fragments into one construct

Guihong Tan, Change Tan

Division of Biological Sciences, University of Missouri-Columbia, MO 65211, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Materials
3.2. Preparation of DNA fragments
3.3. Yeast transformation
3.4. Yeast plasmid rescue
4. Results
4.1. Principle of split-marker-mediated multiple-piece cloning
4.2. Developing of split-marker-mediated multiple-piece cloning
4.3. Comparison of split-marker-mediated versus intact-marker-mediated screening
4.4. Direct usage of raw PCR products and restriction enzyme digestion products
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

We describe a simple method, split-marker-mediated multiple-piece cloning (SMC), to rapidly assemble multiple DNA fragments into one construct in yeast. In this approach, a selectable marker is split into two non-functional, overlapping halves, of which one half is on the plasmid backbone. Homologous recombination reconstitutes the marker gene and assembles all DNA fragments in the desired order. This method allows rapid one-step fusion of various DNA fragments that contain ~30 base pair overlaps in yeast using raw PCR and/or restrict enzyme-digested products. We assembled seven DNA fragments into one contig in a single step by SMC in eight days.