Representational difference analysis (RDA) is a powerful technique for cloning the differences between genomes, and has recently been adapted for cloning differentially expressed genes. RDA, like other PCR-based differential screening methods, is prone to the production of false positives. We have identified a major source of false positives in RDA of cDNA and have introduced improvements which...

We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PC...

A new method for specific reamplification of DDRT-PCR products is presented. After transient ligation of the primary DDRT-PCR fragments into a T-vector, the cDNAs of interest were reamplified by hemi-nested PCR and thermally asymmetric cycles. In contrast to the originally described protocol, this method of reamplification is specific, sensitive, reproducibly gives a high yield of DNA and allow...

Methodology: We tissue cultured the PVX positive potato plants for viral RNA extraction. Total RNA was converted to cDNA for priming CP gene in PCR for amplification. To get the complete sequence of gene, we cloned CP gene into pTZ57R/T cloning vector. Upon double digestion of recombinant plasmid with EcoRI and HindIII restriction enzymes, 710 bp fragment was obtained which confirmed cloning. R...

Iota toxin is produced by Clostridium perfringens type E. This toxin causes antibiotic-associated enterotoxemia in lambs and calves. Iota toxin is a binary toxin that has two components including Ia (the enzyme component) and Ib (the binding component). Ib binds to the surface receptor of target cells and translocate Ia into the cytosol of cells. The aim of this study was to clone toxigenic epi...

Clostridium septicum a Gram positive anaerobic bacterium produces several toxins including alpha, beta, gamma and delta. C. septicum alpha toxin is lethal and is responsible for a serious disease known as gas gangrene. The aim of the present study was to molecular cloning and sequencing of C. septicum vaccine strain alpha toxin gene. Genomic DNA was extracted using standard phenol and chlorofor...

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the ni...