Ultra-Low Background DNA Cloning System

K Goto ,Yukio Nagano

doi:10.1371/journal.pone.0056530

Abstract

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an “ultra-low background DNA cloning system” on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Ampr). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Ampr 5′ UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Ampr 3′ UTR. This cassette allowed conversion of the Ampr-containing vector into the yeast/E. coli shuttle vector through use of the Ampr sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific “origins of replication” to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.

Highlights

DNA cloning is a fundamental technique for modern molecular biology
This method requires a DNA fragment of interest to be PCR-amplified with more than 20 bp of homology to the crossover region of the plasmid vector, and the vector which is digested with a restriction enzyme within the crossover region [1,4]
Multifragment DNA cloning and cloning of large DNA fragments are well-known advantages of yeast-based in vivo cloning, this flexibility can be extremely useful for routine laboratory experiments

Summary

Introduction

DNA cloning is a fundamental technique for modern molecular biology. DNA cloning methods are often inefficient; it is important to develop a more effective method. Yeast-based in vivo cloning is based on homologous recombination and is an efficient cloning method [1,2,3] This method requires a DNA fragment of interest to be PCR-amplified with more than 20 bp of homology to the crossover region of the plasmid vector, and the vector which is digested with a restriction enzyme within the crossover region [1,4]. This method is used in various applications: multifragment DNA cloning [2,3,5,6], cloning of large DNA fragments [7], cloning of targeted regions from eukaryotic chromosomes (transformation-associated recombination (TAR) cloning) [8], and one of the procedures used to synthesize a bacterial cell [9] In this method, the required restriction ends can be located anywhere within the crossover regions of the vector; that is, the sites at which homologous recombination occurs are not always located at the restriction ends in the plasmid vector, making the cloning method more flexible. Multifragment DNA cloning and cloning of large DNA fragments are well-known advantages of yeast-based in vivo cloning, this flexibility can be extremely useful for routine laboratory experiments

Methods

Results

Conclusion