Single-channel multiplexing without melting curve analysis in real-time PCR.

Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target.

Overexpression of GmAKR1, a stress-induced aldo/keto reductase from soybean, retards nodule development.

Development of symbiotic root nodules in legumes involves the induction and repression of numerous genes in conjunction with changes in the level of phytohormones. We have isolated several genes that exhibit differential expression patterns during the development of soybean nodules. One of such genes, which were repressed in mature nodules, was identified as a putative aldo/keto reductase and thus named Glycine max aldo/keto reductase 1 (GmAKR1).

An atypical soybean leucine-rich repeat receptor-like kinase, GmLRK1, may be involved in the regulation of cell elongation.

A leucine-rich repeat receptor-like kinase (LRR-RLK) encoded by one of the genes highly expressed in a specific stage of soybean seed development, referred to as GmLRK1, was identified and characterized. Examination of its kinase domain indicated that GmLRK1 may be a catalytically inactive atypical receptor kinase. An autophosphorylation assay confirmed that GmLRK1 is incapable of autophosphorylation in vitro.

Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers.

Mutations in the YMDD motif of the hepatitis B virus (HBV) polymerase gene increase lamivudine resistance of HBV, highlighting the clinical importance of accurate and sensitive detection of HBV mutants. Using dual-priming oligonucleotide primer technology, an assay that can detect mutations at codons 180 (L528M) and 204 (YVDD, YIDD, and YSDD) by a single-step multiplex PCR was developed. This Seeplex Lami-DR assay was sufficiently sensitive to detect 10(3)HBV/ml and was able to detect minor mutants comprising as little as 2% of the viral population.

Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker.

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots.

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase.

Induction of a homeodomain-leucine zipper gene by auxin is inhibited by cytokinin in Arabidopsis roots.

Homeobox genes are essential regulators of the development of plants as well as other organisms. We chose eight putative Arabidopsis homeobox genes not previously characterized and examined their expression in response to treatment with auxin/cytokinin. One of them, ATHB53, was further studied because it was auxin-inducible and its induction was inhibited by cytokinin.

Annealing control primer system for identification of differentially expressed genes on agarose gels.

We developed GeneFishing technology, an improved method for the identification of differentially expressed genes (DEGs) using our novel annealing control primer (ACP) system. Because of high annealing specificity during PCR using the ACP system, the application of the ACP to DEG discovery generates reproducible, authentic, and long (100 bp to 2 kb) PCR products that are detectable on agarose gels. To demonstrate this method for gene expression profiling, Gene-Fishing technology was used to detect genes that are differentially expressed during development using total RNAs isolated from mouse conceptus tissues at 4.

Annealing control primer system for improving specificity of PCR amplification.

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms.