SOMA: A Single Oligonucleotide Mutagenesis and Cloning Approach

Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.     

Site factors are more important than management for indicator species in semi-natural grasslands in southern Sweden

Plant Ecology - Management of semi-natural grasslands is essential to retain the characteristic diversity of flora and fauna found in these habitats. To maintain, restore or recreate favourable...     

Identification of copy number variants in children and adolescents with autism spectrum disorder: a study from Turkey

Molecular Biology Reports - Copy number variants (CNVs) play a key role in the etiology of autism spectrum disorder (ASD). Therefore, recent guidelines recommend chromosomal microarrays (CMAs) as...     

Exonuclease Degradation of DNA Studied by Fluorescence Correlation Spectroscopy

Abstract

Here we developed an accurate method for kinetic analysis of enzymatic degradation processes of double and/or single-stranded DNA/oligonucleotides using fluorescent reporter dyes. 217-bp DNA fragments were produced by polymerase chain reaction and cleaved by the 3′ to 5′ exonuclease activity of T7-DNA polymerase. The analysis of the products was performed by Fluorescence Correlation Spectroscopy measuring autocorrelation amplitudes and diffusion times. We give proof of (i) complete enzymatic degradation, (ii) retardation of complete enzymatic degradation by internally labelled Rhodamine-4-nucleotides and Cy5-nucleotides, respectively. Data evaluation by global analysis indicated first-order reaction kinetics with full-length DNA and free fluorescent nucleotides in the time window of measurements used.