BACKGROUND Widely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired. RESULTS This paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolec...

Here we report a new methodology to study trace amounts of DNA of unknown sequence using a two-step PCR strategy to amplify and clone target DNA. The first PCR is carried out with a partial random primer comprised of a specific 21-nucleotide 5' sequence, a random heptamer, and a 3' TGGC clamp. The second PCR is carried out with a single 19-nucleotide primer that matches the specific 5' sequence...

Replication studies on human immunodeficiency virus 1 (HIV-1) rely on a few laboratory strains that are divergent from dominant HIV-1 subtypes in the epidemic. Several phenotypic differences between diverse HIV-1 isolates and subtypes could affect vaccine development and treatment, but this research field lacks robust cloning/virus production systems to study drug sensitivity, replication kinet...

PREMISE OF THE STUDY An efficient alternative strategy to conventional cloning was needed to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies. This method would facilitate studies and minimize technical problems typically encountered in cloning methodologies. METHODS We tested a variety of single-strand conformation polymorphism (SSCP) protocols...

PREMISE OF THE STUDY Positional cloning in combination with phenotyping is a general approach to identify disease-resistance gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combined strategy to improve the identification of disease-resistance gene candidates. METHODS AND RESULT...

a highly efficient cloning vector was constructed for cloning pcr products by inserting an 80 bp dna fragment into pgem-5zf (+) vector. the xcm i digestion of this vector gave rise to a 3’ overhanging deoxythymidine offering the possibility of cloning pcr products with 3' adenosine overhang created by taq dna polymerase. furthermore, two ecor i sites were added to the construct for identificati...

background: tuberculosis (tb) remains as a major cause of death around the world. construction of a new vaccine against tuberculosis is an effective way to control it. several vaccines against this disease have been developed. the aim of the present study was to cloning of tb10.4 gene in pcdna3.1+ plasmid and evaluation of its expression in eukaryotic cells. methods: firstly, tb10.4 fragment wa...