Purified DNA functionality

RAPD marker. Oligonucleotide was used OPA-10-⁵′- GTGATCGCAG-³’ (^{Pan et al., 1996}). The volume of the PCR reaction was 25 μL containing 1 X reaction buffer, 1 X, 1.5 mM de MgCl₂, 1 μM of each oligonucleotide, 100 μM of dNTPs, 1.25 U of DNA polymerase (BioTecMol) and 50 ng of genomic DNA. Amplification conditions for the reaction were carried were based according to ^{Chen et al, 2011}, with some modifications. Initial denaturation at 94 °C-4 min, followed by 45 cycles of 94 °C-1 min, 36 °C-1 min and 72 °C-2 min denaturation, alignment and extension, respectively, and a final extension at 72 °C-7 min. The PCR products were separated by horizontal electrophoresis in agarose gels 1.5% (p/v) with TAE buffer stained with ethidium bromide (10 mg/mL).

TRAP marker. The game was used oligonucleotide Arbi 3 ⁵ ́-GACTGCGTACGAATTTGA-³ ́ and SuSy -⁵′-GGAGGAGCTGAGTGTTTC-³’ (^{Hu and Vick, 2003}; ^{Devarumath et al., 2013}). The PCR reaction was performed in a volume of 25 μL containing reaction buffer 1 X, 1.5 mM de MgCl₂, 1 μM of each oligonucleotide, 100 μM of dNTPs, 1.25 U of DNA polymerase (BioTecMol) and 50 ng of DNA genomic. Amplification conditions for the reaction were as follows: initial denaturation at 94 °C-2 min, followed by 5 cycles of 94 °C-45 s, 35 °C-45 s and 72 °C-1 min denaturation, alignment and extension, respectively; followed by 35 cycles of 94 °C-45 s, 50 °C-45 s and 72 °C-1 min denaturation, alignment and extension, respectively. Final extension was 72 °C-7 min (Hu and Vick, 2003). The PCR products were separated by horizontal electrophoresis in agarose gels 1.5% (p/v) with TAE buffer and stained with ethidium bromide (10 mg/mL).

Statistical analysis. The yield (mg/g) and purity (A_260:280and A_260:230) were analyzed for DNA analysis of variance using a completely randomized design factorial 2 (protocols) x 2 ((+N₂ and -N₂) with, giving a total of 4 treatments and 10 repetitions. Experimental unit was considered as a sample of DNA from sugar cane. Test Duncan's multiple range was used to compare mean values between variables than 95% probability using the SAS statistical package. The integrity and functionality of DNA were visually assessed with agarose gels.

DNA yield

The effect of using N₂ performance on DNA obtained from leaves of sugarcane shown in Table 1. The average values of extracted DNA yield +N₂ (0.68 mg/g), were significantly higher than those obtained without the use of the same (0.50 mg/g) (p˃ 0.05). These results exceed those reported by other authors, who extracted DNA from sugar cane, without the use of N₂ and with yields of DNA below 0.30 mg/g of leaf tissue (^{Honeycutt et al., 1992}; ^{Hossain et al., 2006}; ^{Vaze et al., 2010}).

Table1 Performance DNA extracted from young leaves of S. officinarum (+N₂= with liquid nitrogen; -N₂= no liquid nitrogen). 

The results obtained in this study indicate that the use of N₂ increased performance S. officinarum DNA. Generally, the use of N₂ is widely documented in plants (^{Ahmad et al., 2004}; ^{Niu et al., 2008}; ^{Arif et al.,2010}; ^{Sharma et al., 2010}; ^{Cingilli-Vural y Dageri, 2011}; ^{Doosty et al., 2012}; ^{Huaqiang et al., 2013}a; Huaqiang et al., 2013b); fungi (Niu et al., 2008; ^{Motkova y Vytrasova, 2011}; ^{Caceres et al., 2012}; ^{Prabha et al., 2013}) and animals (^{Gross-Bellard et al., 1973}; ^{Chen et al., 2010}; ^{Eschbach, 2012}). Using N2 freezes -196 to -210 °C tissue samples, facilitating all ruptured cell walls and membranes, allowing exposure of nucleic acids with extraction buffer (^{Caceres et al., 2012}).

This suggests that the use of N₂ probably releases a larger amount of nucleic acids of a larger number of cells, yielding improved performance. Also reported obtaining DNA of 2012). These results suggest that the use of spermidine and PEG is not necessary for DNA quality S. officinarum, reducing the cost of the extraction process.

Moreover, ^{Aljanabi et al. (1999)}, obtained high yields of DNA from 0.50 to 0.80 mg/g tissue meristem sugarcane using the "Ultra-Turax" equipment for homogenization of samples and a modification of the CTAB method of ^{Doyle and Doyle (1990)} which included high concentrations of CTAB (20%) and NaCl (6 M) to remove polysaccharides. In this paper the methodology described by the aforementioned author (Protocol II), using 100 mg of leaf tissue (not meristems) was reproduced. The DNA yields obtained were very low and remained in a range of 0.10 to 0.30 mg/g. These results indicate that the type of fabric used and equipment for the extraction of DNA from S. officinarum could explain the differences between the yields obtained with the two protocols. Therefore, it is not necessary to use high concentrations of CTAB and NaCl to obtain high purity DNA from young leaves of S. officinarum.

^{Hossain et al. (2006)} evaluated three DNA extraction protocols for sugar cane from leaves and meristems, with a change in the methodology proposed by ^{Aljanabi et al. (1999)}. The authors replaced the N-lauryl sarcosine sodium dodecyl sulfate by (SDS), both anionic detergents which break the covalent bonds between proteins, denaturing the DNA purification process. The authors obtained a yield of DNA of 0.04 mg/g, significantly lower the here reported for leaves (0.20 mg/g) (Table 1) using the protocol II values. The use of different anionic detergents may have different effectiveness on the breaking of covalent bonds of proteins, so probably the use of N-lauryl sarcosine could increase yields DNA of S. officinarum protocol II. Moreover, ^{Vaze et al. (2010)} reported low yields DNA 0.02-0.10 mg/g dry leaf tissue of sugarcane.

These variations between yields can be explained by the type and condition of tissue used, as in the present study used young leaves, which are in constant cell division, so there are as many cells per gram of tissue; addition, the concentration of phenolic compounds, polysaccharides and secondary metabolites is lower (^{Jobes et al., 1995}). ^{Honeycutt et al. (1992)} used mature leaves, where the concentration of phenolic compounds, polysaccharides and secondary metabolites is increased and the number of cells per gram of tissue is less compared to young leaves. Moreover, ^{Aljanabi et al. (1999)} included sugar cane meristems, which have similar properties to the young leaves. Meanwhile, ^{Hossain et al. (2006)} made use of meristems and leaves; while ^{Vaze et al. (2010)} extracted the DNA from dried leaves of cane sugar cane, where the yield and quality of DNA are low and can be degraded. The above results suggest the use of young leaves of S. officinarum extract and purify DNA for high throughput and acceptable purity.

Purity and integrity of DNA

The effect of the use of N₂ and DNA extraction protocols on purity (A_260:A280 and A_260:A230) of the DNA obtained from sugar cane leaves shown in Table 2 and 3. A pure sample of DNA has a ratio A_260:A280 between 1.8-1.9, while the A^260:A230 should be 1.8-2.2 (^{Gallagher and Desjardins, 2011}). The purities (A_260:A280) +N₂ and -N₂ were 2.01 and 2.00, respectively, so no significant difference with this variable (p˃ 0.05) were detected. Moreover, highly significant differences were detected using protocols (p˃ 0.05) (Table 2), while the protocols *N₂ interaction was significant (p˃ 0.05). These results are consistent with those reported in sugar cane ^{Aljanabi et al. (1999)} and ^{Vaze et al. (2010)}, while ^{Honeycutt et al. (1992)} and ^{Hossain et al. (2006)} reported purities below 1.8, so that DNA is probably slightly contaminated by proteins and/or phenols (^{Gallagher and Desjardins, 2011}; ^{Huaqiang et al., 2013a}).

Table 2 Comparison of purity (A_260:A280 nm) of DNA extracted from S. officinarum (+N₂= with liquid nitrogen; -N₂= no liquid nitrogen). 

Table 3 Comparison of purity (A_260:A230 nm) of DNA extracted from S. officinarum (+N₂= with liquid nitrogen; -N₂= no liquid nitrogen). 

^{Aljanabi et al. (1999)} obtained for all samples A_260/280 of between 1.76-1.96 and reported that the RNA was degraded in the extraction process, however, in this study using the same methodology (Protocol II), was reported for all samples values of the ratio A_260/280 above 2 (Table 2), indicating the presence of RNA, which was not degraded during the extraction process as described by ^{Aljanabi et al. (1999)}, so DNA quantifications II protocol could be overestimated. This was confirmed by electrophoresis on an agarose gel (Figure 1, A). These results suggest the use of RNase A in the DNA extraction process. Using the protocol I RNase A was not eliminated, this suggests that it may be possible omission of incubation at 37 °C and subsequent deactivation and cleaning of this enzyme, saving time. It has not been observed that there is inhibition effect of RNase A in the PCR reactions, so the inclusion of RNase A (10 mg/mL) in the final resuspension of DNA is recommended.

Figure 1 Gel electrophoresis in agarose genomic DNA of S. officinarum extracted with two protocols based on the CTAB method from leaf tissue. He was loaded in each lane 1 μg of DNA. MPM= molecular weight marker, 1 Kb Plus (Invitrogen)A) +N₂ extraction. Protocol I= lanes 1-9; protocol II= lanes 10-18. B) Removal -N₂. Protocol I= lanes 1-4; protocol II= lanes 5-8.  

The purities (A_260:A230) +N2 and -N2 being 1.78 and 1.93 were respectively detected with this variable significant differences (p˃ 0.05), while the effect of the protocols was highly significant (p˃ 0.05) and interaction protocols *N₂ was significant (Table 3). Data reported to the protocol I indicated that the DNA is free polysaccharides. The DNA extraction methods specific for S. officinarum described by ^{Honeycutt et al. (1992)}, ^{Aljanabi et al. (1999)}, ^{Hossain et al. (2006)} and ^{Vaze et al. (2010)} do not report the ratio A_260:A230 as second criterion of purity of DNA, to which the polysaccharides are detected. In the protocol II showed lower values of A_260:A230 of the 1.8 (Table 3), so probably the DNA are slightly contaminated with polysaccharides or phenols. I use +N₂ protocol is recommended to extract DNA from leaves of S. officinarum as both criteria of purity (A_260:A280 and A_260:A230) they were optimal.

The integrity of genomic DNA influenced by the use of N₂ and the two extraction protocols in young leaves of sugarcane in Figure 1. Agarose gels with DNA extracted from leaves were ground with N₂ showed banding patterns genomic DNA of high molecular weight in all lanes with two protocols evaluated (Figure 1-A), so that no degradation of the genomic DNA was detected. The protocol II samples (Lanes 10-18) RNA showed contamination due to the omission of treatment with RNase A as described by ^{Aljanabi et al. (1999)}. The protocol I turned out to be the best for full DNA without RNA residues (Figure 1-A, lanes 1-9). The genomic DNA obtained is similar integrity to that reported by ^{Aljanabi et al. (1999)} and ^{Vaze et al. (2010)} in sugarcane and other authors who used the method of CTAB in cowpea (^{Huaqiang et al., 2013a}; ^{Huaqiang et al., 2013b}). Moreover, the DNA extracted DNA patterns showed -N₂ scanning using the two protocols (Figure 1-B), indicating degradation. Similar results were obtained by ^{Honeycutt et al. (1992)}. The use of N₂ is recommended to extract DNA with acceptable integrity S. officinarum leaves.

Functionality

In the Figure 2 shows the profiles of two molecular markers used to verify the performance of isolated DNA protocols set out in this work. The RAPD marker for all samples amplified perfectly without any effect by the variables N₂ and protocols (Figure 2-A) is noticed. Moreover, waste of RNA (Figure 1, A) protocol II had no negative effect on the PCR reaction, a good profile of amplified bands. Moreover, with the marker pattern TRAP bands with better quality samples they were treated +N2 was obtained (Figure 2-B, lanes 1 and 2). The extracted samples -N₂ showed a pattern of diffuse bands and poor quality for interpretation (Figure 2-B, lanes 4 and 5). It is likely to raise the profile of bands with changing the annealing temperature (Tm) of the oligonucleotide. Moreover, considering that in RAPD markers is complicated reproducibility in other labs, it is important that the DNA be optimal values of A _260:280 and A_260:230 to over come this problem; so the use of the protocol I +N₂ is recommended to extract DNA from S. officinarum sheets for use with molecular markers.

Figure 2 Molecular markers with the DNA of S. officinarum leaves extracted with two protocols based on the CTAB method. MPM= molecular weight marker 100 bp (Invitrogen). A) RAPD with oligonucleotide OPA 10. B) TRAP with oligonucleotide SuSy and Arbi 3. Lanes 1 and 2= ground +N2 protocols I and II, respectively samples. Lanes 4 and 5= ground samples -N2 protocols I and II, respectively. 

In addition to the molecular markers used to explore the genetic diversity of S. officinarum, the DNA obtained with I +N₂ protocol has been successfully used in PCR-based methodologies and sequencing for detection of pathogens in sugar cane: Xanthomonas albilineans, Leifsonia xyli subsp. xyli, Puccinia melanocephala and P. kuehnii cause diseases of economic importance: scald, rickets, rust brown and orange, respectively rust. It has also been used in real time PCR for detection of Candidatus Liberibacter asiaticus, causative agent of Huanglongbing in several species of citrus (Citrus aurantifolia, C. limon y citranges) (data not shown).