Abstract
BackgroundMany established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems.ResultsWe designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley.ConclusionTo facilitate high-throughput linkage mapping and other genomic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler) ecotypes.
Highlights
Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids
We have found that one of the variant extraction buffers, hereafter called Sucrose Solution, exhibited no change in the efficiency of PCR amplification judged by ethidium bromide-stained agarose gels in response to varying the pH from 7.0 to 8.0, or changing the salt and sucrose concentrations between 200 to 400 mM NaCl and 300 to 440 mM sucrose, respectively
We have demonstrated above that plant extracts prepared by the Sucrose Prep are suitable for RT-PCR assays (Figures 5 and 6)
Summary
Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Several EMS (ethylmethansulfonate) and T-DNA mutagenized populations offer valuable genetic resources for wide-scale functional genomics studies in A. thaliana [2,3,4,5,6]. These studies require high-throughput DNA and RNA isolation from tens to thousands of plants. Linkage mapping strategies in A. thaliana are still restricted by the number of known polymorphisms available between various ecotypes, such as Col-0 and Ler. The frequent use of segregating populations derived from Col-0 × Ler crosses, especially in the study of flowering time and plant development, would significantly benefit from a larger set of well-characterized and tested markers. To develop a facile map based cloning approach, we refined the current design of polymorphic markers such that all polymorphic markers can be resolved on 3% (w/v) agarose gels and detected by ethidium bromide staining
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