BMC Biotechnology BioMed Central Methodology article

Background After transformation, plants that are homozygous and contain one copy of the transgene are typically selected for further study. If real-time PCR is to be used to determine copy number and zygosity, it must be able to distinguish hemizygous from homozygous and one-copy from two-copy plants. That is, it must be able to detect two-fold differences. Results When transgenic Nicotiana attenuata plants which had been previously determined by Southern analysis to contain one or two copies of the transgene, were analyzed by real-time PCR (2-ΔΔCt method), the method failed to confirm the results from the Southern analysis. In a second data set we analyzed offspring of a hemizygous one-copy plant, which were expected to segregate into three groups of offspring in a 1:2:1 ratio: no transgene, hemizygous, homozygous. Because it was not possible to distinguish homozygous from hemizygous plants with real-time PCR, we could not verify this segregation ratio. Conclusions Detection of two-fold differences by real-time PCR is essential if this procedure is to be used for the characterization of transgenic plants. However, given the high variability between replicates, a detection of two-fold differences is in many cases not possible; in such cases Southern analysis is the more reliable procedure.


Background
Molecular biological studies of plants require high-quality DNA. Several DNA extraction procedures for isolating genomic DNA from various plant sources have been described, including the salt extraction method and the cetyltrimethyl ammonium bromide (CTAB) method [1] and its modifications [2,3]. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed.
Most methods require the use of liquid nitrogen [4] or freeze-drying (lyophilization) [5,6] of tissue for the initial grinding, and these processes are unavailable in many regions of the world. After grinding the tissues in various extraction buffers, DNA is extracted with phenol-chloroform, or the extract is dialyzed against EDTA and a buffered Tris-HCl solution [7]. After extraction, the aqueous phase is concentrated, either by ethanol or isopropanol precipitation [8,9], or with microconcentrators (e.g., the Wizard genomic DNA purification system; Promega, USA). However, these methods are not time efficient for consistently obtaining PCR-quality DNA from calluses and plants, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc.
In our laboratory, we investigate the stability of transgenes expressed in calluses or plants transformed by nuclear or chloroplast transformation in tobacco, lettuce, potato, etc. In addition, we need high-quality genomic DNA for Southern blot analysis to confirm homologous recombination in chloroplast transformation [10]. For our purposes, we desire a simple and fast procedure for obtaining plant genomic DNA for PCR, and good-quality DNA for complete enzyme digestion for Southern blot analysis. Therefore, we present a protocol for extracting genomic DNA from fresh calluses and plant leaves that is applicable to a variety of organisms, regardless of the complexity of their genomes. In addition, we present a rapid and reli- (A) Lane 1, the DNA template was the supernatant from the first phenol:chloroform:isoamyl alcohol extraction (protocol 1); lane 2, the DNA template was the supernatant after two phenol:chloroform:isoamyl alcohol extractions and one chloroform extraction (protocol 2); lane 3, the DNA template was prepared with an additional ethanol precipitation (protocol 3). (B) PCR products amplified using only the DNA template from protocol 1. 1 kb, DNA molecular weight ladder.
able procedure for extracting genomic DNA for PCR or Southern blot analysis from a small amount (~0.5 cm 2 ) of leaf tissue.

Results and discussion
We describe a simple and reproducible procedure for RAPD or PCR amplification of transgenes from various plant sources. Three different variations of the genomic DNA extraction protocol for RAPD analysis were compared. After simple plant leaf and callus tissue homogenization with DNA extraction buffer using a hand-operated homogenizer, the leaf and callus cells were lysed with 20% SDS. Then, genomic DNA was extracted with the same volume of phenol/chloroform/isoamyl alcohol (25:24:1). An aliquot of the supernatant (~5 µl) was diluted 5 fold with sterile dH 2 O, and PCR was performed using 1 µl of the diluted supernatant as a template ( Figure  1, lane 1). Alternatively, after phenol/chloroform/isoamyl alcohol (25:24:1) extraction, the supernatant was transferred to a fresh tube for a second phenol/chloroform/isoamyl alcohol (25:24:1) extraction followed by a chloroform extraction. An aliquot of the supernatant (~5 µl) was diluted 5 fold with sterile dH 2 O, and PCR was performed using 1 µl of the diluted supernatant as the DNA template ( Figure 1, lane 2). In the third variation, after chloroform extraction the supernatant was transferred to a fresh tube and precipitated with two volumes of ethanol. After washing the DNA pellet with 70% ethanol, the DNA pellet was dissolved in 50 µl of sterile dH 2 O containing 20 µg ml -1 DNase-free RNase A. For PCR, 50 ng of the DNA were used as the template (Figure 1, lane 3).
DNA samples prepared using the three different extraction procedures (lanes 1, 2, and 3 in Figure 1) were subjected to PCR amplification using two 10-mer random primers: RAPD-1 and RAPD-2 (Genotech, Korea) ( Figure 1). All the genomic DNA samples produced a clear, sharp, and reproducible PCR product when primer RAPD-1 was used for PCR amplification ( Figure 1A). Although three variations of the DNA extraction procedure were used, there was little difference between lanes 1, 2, and 3. Only a difference in the intensity of the band was observed, which may be due to the different template concentrations used for the PCR reaction. This result suggests that the supernatant after the first phenol treatment (protocol 1) was sufficiently pure to be used as the DNA template for PCR amplification. Therefore, PCR amplification with another random primer, RAPD-2, was performed using the DNA template extracted using the simplest protocol ( Figure  1B). The PCR amplification was successful, and the same banding pattern was seen when we repeated the PCR amplification. Therefore, we confirmed that the DNA template extracted using the simplest method was sufficient for RAPD, and it was used as the DNA template to amplify specific DNA or transgenes from transgenic calluses or plants.
To examine the presence of bar [11,12] or the LTB gene [13] at a directed site in the chloroplast DNA after homologous recombination in transplastomic tobacco plants, putative transformants were screened by PCR analysis (Figure 2). PCR amplification using primer combinations Bar-F/Bar-R, 1-F/1-R, and LTB-F/LTB-R resulted in 550-, 1700-, and 380-bp fragments, respectively. Primers 2-F/2-R produced 2200-or 1900-bp fragments containing bar and LTB, respectively, which confirmed the site-specific integration in the chloroplast genome (Table 1). No detectable product was produced using genomic DNA from wild-type plants ( Figure 2B, lane 1), demonstrating the specificity of these primers and genomic DNA extracts. Therefore, we concluded that chloroplast DNA was also amplified, although we did not use liquid nitrogen, but simply used a hand-operated homogenizer with a plastic tip. We also successfully amplified specific foreign genes from transgenic tobacco plants transformed using the nuclear transformation method, including the α-interferon (550 bp) [14], the core epitope of the PEDV gene (420 bp) [15,16], the LTB gene (380 bp) [17], and the A plus B subunit of the Helicobacter pylori urease gene (2450 bp) [18] (Figure 3). Specific PCR amplification was also conducted using transgenic calluses as well as transgenic plants. In transgenic calluses derived from Siberian ginseng plants, α-interferon was successfully amplified, showing a 580-bp fragment in 1% agarose gels.
Using the third protocol, the DNA concentrations obtained were between 20 and 30 µg/0.5 cm 2 plant leaf, and the absorbance ratios (A 260 /A 280 ) were between 1.7 and 2.0. However, the DNA concentrations from rice, maize, and poplar were relatively low (< 3 µg). This may be because homogenization using a hand-operated homogenizer with a plastic tip is incomplete, since the leaves of these plants are stronger than the leaves of tobacco, potato, cabbage, lettuce, and Siberian ginseng. Genomic DNA from various plant sources was electrophoresed on 1% agarose gels, and high-molecular-weight DNA was obtained ( Figure 4A). When the genomic DNA was digested with EcoRI and HindIII, the DNA was completely digested, and could be used for Southern blot analysis. Therefore, we concluded that the purity and quality of the genomic DNA was sufficient for enzyme digestion.
There are many advantages in using our genomic DNA extraction method to obtain template for PCR amplification. Many different plants could be amplified using the same DNA extraction method and the same PCR protocol. Using this protocol, we successfully amplified DNA repeatedly from all eight plant sources examined. Our procedure is not only very simple, but is also time and cost effective. After homogenization in DNA extraction buffer using a hand-operated homogenizer, the template DNA for PCR could be extracted by phenol/chloroform/isopropyl alcohol treatment. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. The time required for our DNA extraction method is less than 30 min, which is extraordinary compared with other genomic DNA extraction methods. With our procedure, leaf tissue (~0.5 cm 2 ) is put in a 1.5-ml microfuge tube and homogenized directly; consequently, a very small sample is required for DNA extraction. There is no sample waste with our method, whereas much larger samples are required when plant samples are ground in a mortar and pestle with liquid nitrogen and transferred to a tube. Previously reported techniques require several steps [19], use of expensive enzymes such as proteinase K [20], or beads and shakers [21]. Although the protocol for one-step plant DNA isolation was developed by Burr et al. [22], if plant material more than 1 mm 2 was used in the extraction, co-extracts (e.g., chlorophyll) were extracted alongside the DNA and inhibited the PCR. On the contrary, our protocol does not require appropriate sample size to extract DNA. Warner et al. [23] also reported a rapid DNA extraction method in barley, which requires NaOH. However, the extracted DNA samples were easily degraded. The DNA samples extracted by our protocol were very stable and could be stored for a long time without degradation.
We find the new method very useful in our laboratory, since limited transgenic plant tissue or callus is sometimes available in a culture bottle. Therefore, the simplicity, efficiency, speed, and lack of a requirement for expensive facilities make our method an attractive alternative to existing methods of genomic DNA extraction.

Conclusions
Our objective was to extract genomic DNA with a simple and fast procedure for PCR and enzyme digestion. The present protocol is for extracting genomic DNA from fresh calluses or plant leaf tissues that is applicable to a variety of organisms, regardless of the complexity of their genomes. Our procedure is not only very simple, but is also time and cost effective. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. In addition, a very small sample is required for DNA extraction.

Plant material
We examined plant material from plant collections commonly used for foreign gene expression: tobacco (Nicotiana tabacum), potato (Solanum tuberosum), cabbage (Brassica oleracea), rice (Oryza sativa), lettuce (Lactuca sativa), maize (Zea mays), poplar (Populus nigra), and Siberian ginseng (Eleutherococcus senticosus). The plants used for genomic DNA extraction were grown in a culture room or greenhouse. Tobacco, potato, cabbage, lettuce, and Siberian ginseng were grown in a culture room. Seeds were surface-sterilized with 70% ethanol for 3 min, and then with 10% sodium hypochlorite for 15 min. The seeds were washed five times in sterile water and placed in Petri dishes containing 4.6 g l -1 MS salts [24], 30 g l -1 sucrose, and 7.5 g l -1 bactoagar at pH 5.7. The seeds were grown in a controlled environment at 25°C on a 16-h continuous light and 8-h dark daily cycle. Rice, maize, and poplar plants were grown in a greenhouse for genomic DNA extraction. Transgenic tobacco plants and Siberian ginseng calluses were also used to extract genomic DNA and to confirm foreign gene insertion by PCR amplification.

DNA extraction (Figure 5)
We tested three different variations of the genomic DNA extraction procedure. About 0.5 cm 2 of culture room-or greenhouse-grown plant leaves were put in a 1.5-ml microfuge tube. The leaf tissue was homogenized in 50 µl DNA extraction buffer (500 mM NaCl, 100 mM Tris-HCl pH 7.5, and 50 mM EDTA pH 7.5), using a hand-operated homogenizer (Sigma, Z35997-1) with a plastic pestle, for 15~20 s. After an initial homogenization, another 150 µl of DNA extraction buffer were added and homogenized with the same homogenizer for 15~20 s. Then, 20 µl of 20% SDS were added and vortexed for 30 s. The samples were incubated at 65°C for 10 min for cell lysis. At this point, three different DNA extraction protocols were used for PCR amplification. Protocol 1: An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the samples, mixed by vortexing for 30 s, and then centrifuged at 10,000 g for 3 min at 4°C. The supernatant was diluted 5 fold, and 1 µl of the supernatant was used as the DNA template for PCR analysis. Protocol 2: The supernatant from protocol 1 was transferred to a fresh tube and extracted one more time with phenol/chloroform/isoamyl alcohol (25:24:1) and then with chloroform. The supernatant was diluted 5 fold, and 1 µl of the supernatant was used as the DNA template for PCR analysis. Protocol 3: The supernatant from protocol 2 was transferred to a fresh tube, and a double volume of ethanol was added to each sample, mixed well, and the samples were incubated at -20°C for 30 min. The samples Agarose gel electrophoresis of undigested and digested genomic DNA were then centrifuged at 10,000 g for 10 min at 4°C. The pellet was washed with 70% ethanol, dried, and resuspended in sterile dH 2 O containing 20 µg/ml DNase-free RNase A. The concentration and purity were determined from the A 260 /A 280 ratio using a spectrophotometer. Five micrograms of each genomic DNA sample were incubated at 37°C for 3 h for complete digestion with 20 U of EcoRI and HindIII (Life Technologies, USA) in a total volume of 100 µl and analyzed on 1.0% agarose gels using 15 µl aliquots of the reaction mixture.

Analysis of DNA and PCR amplifications
Five micrograms of each genomic DNA sample measured by spectrophotometer were incubated at 37°C for 3 h for complete digestion with 20 U of EcoRI and HindIII in a total volume of 100 µl and analyzed on 1.0% agarose gels using 15 µl aliquots of the reaction mixture. By using the genomic DNA isolated from the leaves or calluses of wild-type and transgenic plants, PCR amplifications were performed on a Perkin Elmer GeneAmp PCR System 2400 (Biorad, USA) in a total volume of 25 µl containing 1 × PCR buffer, 0.2 mM dNTP, 10 pmol of each primer (Table  1), 50 ng template DNA from plants, and 0.25 U Ex-Taq DNA polymerase (Takara, Japan) using the following profile: a 3-min denaturation at 94°C and 40 cycles of 1-min denaturation at 94°C, 1-min annealing at 37°C for RAPD or 55°C for specific transgene amplification, and a 2-min extension at 72°C, followed by a final extension at 72°C for 7 min. The PCR products were resolved by electrophoresis in 1.0% agarose gels.

Authors' contributions
TJK developed the method and performed majority of the experiments. MSY provided technical assistance, funding and supervision for the work. All authors have read and approved the final manuscript.
Three different DNA extraction protocols for calluses and plants Figure 5 Three different DNA extraction protocols for calluses and plants