Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins

The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology. Nature Methods

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications. The availability of the human genome sequence 1,2 and genome diversity databases 3-5 at the beginning of the 21 st century are causing a paradigm shift away from the standard protocol of medical care toward genotyped medicine. This new type of medicine is based on the accumulating knowledge of gene polymorphisms (SNPs) and their relationship to specific phenotypes, such as disease predisposition, drug metabolism and disease development. A key step for the development of individualized medicine is the ability to rapidly test patients for these SNPs and/or other mutations correlated to diseases and disease predisposition. Supporting this point, the US Food and Drug Administration has required the drug industry to publicly provide SNP data examined in the process of procuring a drug license. Today SNP genotyping technologies 6-9 are still a bottleneck in drug discovery research and clinical applications. But high-throughput gene analysis and SNP detection technologies will inevitably become both cheaper and faster in the future. Besides SNP genotyping, these improved sequence-detection technologies would also allow and advance studies in other disciplines such as population genetics, the global surveillance of infectious disease and the study of somatic mutations in human cancer. Almost all previously developed SNP-detection systems consist of two steps: amplification (usually by PCR) and detection of SNP (using DNA fragments amplified in the first step). This approach is reasonably fast, but to shorten the time required and simplify the detection, it is ideal to develop a one-step method, in which the amplification itself can be the SNP detection signal. The difficulty in developing such a technology is in the suppression of the background amplification. For example, primers for allele-specific primer PCR are designed with the nucleotide mismatch at the 3¢ end of the PCR primers, but the misamplified PCR products primed from mismatched primers are still exponentially amplified, producing background signals that must be addressed. Here we report SMAP 2, the first rapid one-step SNP detection technology in which the amplification of the targeted DNA is the signal of the target SNP itself