Sequencing data and MLPA analysis data in support of the effectiveness and reliability of an asymmetric PCR-Based approach in preparing long MLPA probes.

ABI PRISM 3100 Genetic Analyzer, a multi-color fluorescence-based DNA analysis system with 16 capillaries operating in parallel, was ideal tool both for DNA sequencing and DNA fragment analysis [1,2]. To demonstrate the effectiveness and reliability of an asymmetric PCR-Based approach (X.Y. Ling, G.M. Zhang, G. Pan, H. Long, Y.H. Cheng, C.Y. Xiang, L. Kang, F. Chen, Z.N. Chen, Preparing long probes by an asymmetric PCR-based approach for multiplex ligation-dependent probe amplification (MLPA), Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.03.031, in press) in preparing the long MLPA probes that were generated with a M13-based method before [4], some prepared long MLPA probes were sequenced and then tested in MLPA analysis. Sequencing data shows that the long MLPA probes were identical to the designed ones, indicating the long probes can be easily prepared with the new method, and the MPLA analysis data shows that the results of MPLA analysis with these long probes were as same accurate and specific as with ones prepared with other methods. The sequencing data was not presented in the research article (X.Y. Ling, G.M. Zhang, G. Pan, H. Long, Y.H. Cheng, C.Y. Xiang, L. Kang, F. Chen, Z.N. Chen, Preparing long probes by an asymmetric PCR-based approach for multiplex ligation-dependent probe amplification (MLPA), Anal. Biochem. (2015), 10.1016/j.ab.2015.03.031, in press), but the MLPA analysis data was converted into figure 4 and figure 5 of the research article.

Preparing long probes by an asymmetric polymerase chain reaction-based approach for multiplex ligation-dependent probe amplification.

To clearly discriminate the results of simultaneous screening and quantification of up to 40 different targets-DNA sequences, long probes from 100 to 500 nt, rather than smaller or similar-sized synthetic ones, were adopted for multiplex ligation-dependent probe amplification (MLPA). To prepare the long probes, asymmetric polymerase chain reaction (PCR) was employed to introduce non-complementary stuffers in between the two parts of the MLPA probe with specially designed primers, then restriction enzymes were selected to digest the double-stranded DNAs, and finally polyacrylamide gel electrophoresis was used to purify the single-stranded DNAs (i.e., the long probes). By using this approach, 12 long probes were prepared and used to identify genetically modified (GM) maize. Our experimental results show that the prepared long probes were in full accordance with the designed ones and could be assembled in 4-, 7-, and 10-plex MLPA analysis without losing result specificity and accuracy, showing they were as effective and reliable in MLPA analysis as those prepared with M13-derived vectors. This novel asymmetric PCR-based approach does not need expensive equipment, special reagents, or complicated operations when compared with previous methods. Therefore, our new approach could make MLPA analysis more independent, efficient, and economical.

Antisense expression of mitochondrial ATP synthase subunits OSCP (ATP5) and gamma (ATP3) alters leaf morphology, metabolism and gene expression in Arabidopsis.

Determination of the role of mitochondrial (mt) ATP synthesis in plant metabolism is complicated by chloroplastic ATP synthesis. To differentiate ATP synthesis from these two organelles, we created transgenic Arabidopsis plants in which two different subunits of the mt ATP synthase, the oligomycin sensitivity-conferring protein (OSCP) (=delta) (ATP5) and the gamma (ATP3) subunit, were expressed individually in antisense orientation under the control of a dexamethasone-inducible promoter. The phenotypic effects of antisense expression were identical for both atp5 and atp3. Seedling lethality resulted from induction during germination in the light, demonstrating the essentiality of both gene products. Reduced expression of either gene resulted in stunting of dark-grown (etiolated) seedlings, downward curling or wavy-edged leaf margins of light-grown plants and ball-shaped unexpanded flowers. Antisense induction reduced total ATP levels in dark-grown (etiolated) seedlings germinated on media lacking sucrose, but increased total ATP levels in induced light-grown plants and in induced dark-grown seedlings germinated on media containing sucrose. Induction reduced transcript levels for two transcription factors (TCP3 and TCP4) whose decreased expression is associated with a similar wavy-edged leaf phenotype in Arabidopsis, and increased transcript levels for dynamin-related proteins whose increased expression is associated with increased mt division. Reduced expression of these subunits of the mt ATP synthase is proposed to disturb cellular redox states, which ultimately manifest downstream as diverse and seemingly unrelated phenotypes.