The Sucrose Prep is not unique in being a quick DNA isolation protocol. Kasajima et al.  have exploited the method of Edwards et al.  to develop a rapid method for marker and transgene detection, and have demonstrated amplification of fragments up to 1.4 kb in size. As Langridge et al.  and Petersen et al. , we have also observed that DNA can be extracted by grinding the plant tissue in pure water and transferring a sample aliquot of the extract to a PCR reaction or that DNA templates can be delivered by adding a small amount of tissue to a PCR reaction mixture (data not shown). Therefore, we tried using extremely small amounts of tissue sampled with pipette tips by puncturing leaf tissue and immediately touching the tips into prepared PCR reaction mixtures. This 'Touch-and-Go' method is comparable in PCR amplification efficiency of DNA fragments up to about 1 kb with the Sucrose Prep and other rapid nucleic acid isolation methods. The stability of the Sucrose Prep in providing templates for numerous PCR or RT-PCR reactions however encouraged us to continue optimization of the method in connection with various high-throughput applications.
Alkaline lysis with NaOH [21–23] has also been successfully used in rapid isolation of DNA, however PCR amplification of DNA fragments are typically smaller than 2 kb in size. A common step between the majority of rapid procedures and the Sucrose Prep is the inclusion of a 'boiling' step. Burr et al.  used a thermal cycling protocol from 65°C to 96°C for a total time of 11.5 min, whereas Thomas and Henry  had optimized their protocol for DNA extraction from dried tissue by heating for 10 min at 95°C. Many quick DNA preparation methods dilute out contaminants from the harvested tissue, which interfere with the PCR reaction, by raising the extraction volume [22–26]. Sucrose Prep also employs such a dilution step by using about 50 μl extraction buffer for 2.5 mg tissue. Another component of the Sucrose Solution is the use of high salt, which is also employed in the protocol of Wang et al.  and is a principal component of DNA extraction buffers described by Edwards et al.  and Walbot and Warren .
The Sucrose Prep has been optimized for A. thaliana tissue, but is also suitable for DNA isolation from other species, including tobacco and barley (Figure 8). Therefore, Sucrose Prep should also work for species such as maize, wheat, rice, potato, tomato and other plants that have low to moderate concentrations of phenolics and starch
The combination of bulk segregant analysis with our new polymorphic DNA marker set proved very effective in rapidly locating a mutation of interest within a 10 cM interval. Tracking F2 segregation with the Sucrose Prep dramatically eased the analysis, as we employed a DNA shaker and thermal cycling blocks for heating, making future optimisation with robots possible. DNA sequencing of a candidate gene identified a mutation in the first exon of the PHYB gene, causing a premature stop codon at amino acid 554. The resulting truncated protein is predicted not to contain the PAS domain, which are important in phytochrome function and mediate interaction with putative signalling partners [27–29].
The Sucrose Prep also proved useful in screening for homozygous T-DNA insertion mutants and it was particularly useful for rapid identification of double homozygous knock-out lines that carried T-DNA insertions bearing identical selectable markers (Figure 3 and Figure 5). Occasionally, certain PCR primers did behave differently between conventional DNA preparations and templates obtained by the Sucrose Prep (Figure 1 and Figure 4). Nonetheless, in most cases where a primer combination did not work with Sucrose Prep, they also failed to produce PCR amplification with conventional methods under the aforementioned size limits (data not shown).
Pre-screening of segregating F2 or T2 EMS- and T-DNA-induced mutation populations with the Sucrose Prep greatly reduced the number of lines requiring further characterization. Upon fast screening with Sucrose Prep, detailed analysis always led to the identification of homozygous mutant lines that were confirmed by other DNA and RNA isolation methods.
RNA isolation from plants is often a lengthy process requiring toxic chemicals or expensive kits, and requires a very clean practice due to vast contamination of RNAses. A quick RNA isolation method is therefore highly desirable. We have demonstrated above that plant extracts prepared by the Sucrose Prep are also suitable for RT-PCR assays (Figures 5 and 6). Due to high concentration of DNA in the extracts, the primers must be designed in exons separated by introns in order to distinguish DNA from cDNA (Figure 6). DNA contamination is not a unique problem to our approach but is common to several other RNA isolation methods. The only major disadvantage of our quick RNA isolation method is that the RNA to DNA ratio is very low as compared to other methods where RNA is concentrated through several steps. However, our method is very useful and even superior to other methods in certain applications requiring speed and use of limited amounts of plant tissue. Thus, the method is particularly useful if cells expressing the gene of interest are restricted to certain tissue, such as hydathodes, major and minor veins, emerging young leaves, flower organs such as nectaries, flower abscission zones, sepals petals, anthers, gynoecium, root tips, as well as local pathogen infected tissues and islands of cells generated by transposon jumping. One of the limitations of our method is that it is not suitable for genes that are expressed at very low levels. The 'Touch-and-Go' method is not suitable for quantitative RT-PCR applications, due to the varying amount of RNA isolated during sampling.
Using the Sucrose Prep and the 'Touch-and-Go' methods, we have identified homozygous T-DNA knockouts for the AtWRKY36 and AtWRKY70 genes (Figure 6). With respect to PCR amplification of DNA samples, we found the 'Touch-and-Go' method extremely useful and time saving, especially when screening for the presence or absence of PCR products of less than 500 bp in size. We had however variable success rates if the size of the expected PCR products was larger than 1 kb. As with the Sucrose Prep, the 'Touch-and-Go' method worked very well for tobacco in amplifying DNA fragments up to 2 kb, however, in barley it failed to amplify the largest predicted fragment of 1.7 kb and the amount of amplification product varied for fragments less than 1 kb in length. This difference observed in the reproducibility of DNA isolation between tobacco and barley is possibly due to the lower number of cells that are disrupted in barley plants upon puncturing with the pipette tip. Nevertheless, the simplicity, speed and reproducibility of the 'Touch-and-Go' approach and the robustness and relative speed of the Sucrose Prep method makes them ideal for high-throughput PCR based screens in alternative transgenic approaches replacing the use antibiotic resistance selectable markers .