ABSTRACT
Here we present a simple and convenient sticky/blunt-end ligation method for fusion gene construction. The fusion gene is constructed by seamless ligation of 5'-end phosphorylated blunt ends instead of by overlap extension PCR (OE-PCR). Therefore, the challenge of amplifying large DNA fragments (e.g., the large bifunctional enzyme gene constructed by fusion of two monofunctional enzyme genes) by PCR can be avoided. In addition, synthesis of the inner primers for OE-PCR is not necessary, indicating that this method should be especially convenient for construction of fusion genes with various combinations of multiple fragments (e.g., chimeric gene libraries, fusion gene libraries). As a modification of the commonly used fusion gene construction technique, this method may find a wide range of applications in bioscience and biotechnology.
Subject(s)
DNA Ligases/genetics , DNA Primers/genetics , Polymerase Chain Reaction/methods , Polynucleotide 5'-Hydroxyl-Kinase/genetics , Recombinant Fusion Proteins/genetics , Viral Proteins/genetics , Bacteriophage T4/chemistry , Bacteriophage T4/enzymology , Base Sequence , Cloning, Molecular , DNA Ligase ATP , DNA Ligases/metabolism , DNA Primers/chemical synthesis , Directed Molecular Evolution , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Library , Molecular Sequence Data , Phosphorylcholine , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , Recombinant Fusion Proteins/biosynthesis , Viral Proteins/metabolismABSTRACT
A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.
