A modified protocol for rapid DNA isolation from cotton (Gossypium spp.)

Graphical abstract


Introduction
Extraction of high-quality DNA in sufficient quantity is important for studying the molecular genetics of cotton. However, high endogenous levels of polysaccharides and polyphenols interfere with the isolation of good quality DNA, thereby rendering it unsuitable for downstream analyses [1][2][3]. During cell disruption, phenolic compounds come out of the vacuoles, become readily oxidized and irreversibly bind with nucleic acids and proteins, thus resulting in a dark DNA pellet unsuitable for most enzymatic manipulations [4]. On the other hand, the viscous nature of polysaccharides makes extracted DNA fractious to pipetting; and, it also interferes with various biological enzymes, and especially hinders the PCR reaction by inhibiting the Thermus aquaticus DNA polymerase (Taq. pol) activity [5,6].
Previously reported extraction protocols for cotton are comparatively expensive, time-consuming, require liquid nitrogen or lyophilization and ultracentrifugation in CsCl gradients [2,[7][8][9][10][11]. Although, these methods may yield good quality DNA; however, they are not suitable for the local Pakistani varieties and high-throughput applications, such as restriction fragment length polymorphism (RFLP) and screening of transformants [9,10], that require inexpensive and reproducible DNA extractions. Another major problem for most laboratories in developing countries is the continuous procurement and storage of liquid nitrogen [12]. Over the years to overcome these problems, numerous modifications have been introduced into the original CTAB method [13] to reduce the cost and time of routine DNA isolation [14]; however, none of the modifications have been found to be universally applicable for every plant species due to their chemotypic heterogeneity. Most recent CTAB methods, including this protocol, omit the CsCl gradient ultracentrifugation, selective precipitation steps, use of liquid nitrogen and toxic phenol in favour of a simple, rapid and safe procedure.
In this regard, the described procedure was modified from Paterson et al. [9] to reduce time, cost, and resolve the problems associated with high endogenous levels of secondary metabolites, especially polysaccharides and polyphenols. This method consistently yields high-quality DNA in sufficient quantity suitable for most projects, such as cloning, mapping and marker-assisted plant breeding. An individual can routinely process 24-48 samples and isolate 10-15 mg of high-quality DNA in about 3 h.
In addition, this procedure may also be adapted to a 96-well microplate format [15] for highthroughput DNA extraction.

Plant materials
Seed and leaf tissues were harvested from the cotton variety VH-289 to test the applicability of this procedure. Approximately 3-5 cm 2 (80-100 mg of fresh weight) of the leaf and 100 mg of the seed flour were sampled into 1.5 ml microfuge tubes and placed on ice.  2 Add 400 ml of each buffer A and B into a 1.5 ml nuclease-free microfuge tube containing 100 mg of seed flour. In case of leaf, grind 100 mg of tissue in 500 ml of buffer A using a mortar and pestle, then pour the mixture into a 1.5 ml tube. Pipette 400 ml of buffer B into the same tube.

Reagents and consumables
Note: TissueLyser should be used for high-throughput extraction and to prevent crosscontamination 3 Vortex the mixture for 10 s to mix thoroughly. Incubate the tubes at 60 C for 30 min in a water bath and invert after every 10 min to homogenize.
4 Cool down the tubes at room temperature (RT) and add RNase A (25 mg/ml). Invert the tubes for 4-5 times and incubate at 37 C for 20 min in an incubator.
5 Add 400 ml of CIA and vortex for 5 s to form an emulsion.
6 Centrifuge at 13,000 g for 10 min at RT to separate the organic and aqueous phases.
Note: If the aqueous layer is not transparent then repeat the step 5.
7 Carefully transfer the aqueous (transparent) phase using a micropipette into a new tube.
Note: Wide-bore tips should be used to prevent mechanical damage to DNA.
9 Close the tubes tightly, gently invert for 5-6 times and then precipitate the DNA by incubating at À20 C for 15 min.
Note: To increase the precipitation of the DNA, the tube may be incubated for overnight.
10 Centrifuge at 13,000 g for 5 min to pellet the precipitated DNA.
11 Carefully remove the supernatant without disturbing the pellet and add 400 ml of 70% (v/v) chilled (À20 C) ethanol. Dislodge the pellet by flicking with a finger. 12 Centrifuge at 13,000 g for 5 min and discard the supernatant by decanting. 13 Remove the ethanol residuals by drying the DNA pellet on a heat block.
Note: Do not over dry the pellet because it will make it difficult to dissolve.
14 Dissolve the DNA pellet in 40 ml of nuclease-free water.
Note: TE buffer should be used to dissolve the DNA pellet intended for long storage.

Qualitative and quantitative analyses of the isolated DNA
A simple, rapid and comparatively cheap spectrophotometric analysis was performed to assess the purity of the extracted DNA. Ratios of UV absorption at A 260/280 and A 260/230 were recorded by using a Nano-Drop ND-2000 (Thermo-Scientific, USA). DNA degradation and RNA contamination were assessed through agarose gel electrophoresis.

Results and discussion
The main steps in this method, namely cell disruption, CIA extraction and DNA precipitation, are similar to those described for other plant species [3,9,10,12,14,16,17].
The buffers used in this extraction method have been redesigned to cope with the problems associated with high levels of secondary metabolites. Specifically, the high concentration of phenolbinding reagent (PVP) and NaCl were employed to remove polyphenols and polysaccharides, respectively. Moreover, glucose was used as a reducing agent to avoid contamination and browning of the DNA pellet [18], while βME used as an antioxidant to prevent the oxidation of polyphenols. In addition, to reduce the cost and processing time of the procedure, both buffers A and B were added at same step and RNase A was added before CIA extraction.
Spectrophotometric analysis is one of the most frequently used techniques for quality assessment of a DNA. The ratio of UV absorption at A 260/280 is 1.8 for a pure DNA, any increase in it indicates RNA contamination and conversely, the presence of protein (largely) decreases the value. The recommended absorption ratio at A 260/230 is 2.0-2.22 for impurity free DNA [4]. The mean concentration and quality of the DNA, extracted via this method are presented in Table 1. The values of all extractions were within the accepted range, indicating a low level of contamination.
Being simple and efficient, agarose gel electrophoresis is quite often the method of choice to detect degradation, and impurities e.g. RNA and carbohydrates in a DNA sample [1,19]. Apart from this, DNA, that is susceptible to restriction enzymes and allows PCR amplification, is also considered to be significantly clean DNA. In this regard, the extracted DNA was further subjected to electrophoretic, PCR and restriction analyses. In result, our DNA was found to be highly susceptible to Hind III restriction enzyme digestion (Fig. 1A; lane 2 and 4) and free from degradation and RNA contamination ( Fig. 1A; lane 1 and 3). The PCR (111 bp) product was amplified with the gene (CP4-EPSPS) specific primers and separated on 2% agarose gel (Fig. 1B; lane 3 and 4). In several independent extractions or replicates, reproducible amplification and susceptibility to restriction enzyme digestion were observed.
In conclusion, we described a simple and rapid protocol that can reliably use for routine DNA isolation from cotton and is amenable for high-throughput applications, such as DNA markerassisted selection [20,21] and cloning [22,23]. In addition, this protocol may be used for other plant species that are recalcitrant to other methods due to their high levels of polysaccharides and polyphenols.

Conflicts of interest
None.
Author's contribution QA, AQR, AAS, and TH designed the protocol. QA, IBS and AR carry out the laboratory work and optimize the method. QA wrote the manuscript and all authors revised and approved the final manuscript.