Comparing protocols of DNA extraction from Escherichia coli : Analysis of purity and concentration by gel electrophoresis

Background and objectives: Given the difficulty in establishing an ideal method that can successfully extract bacterial DNA of Gram-negative bacteria, the objective of this work was to compare the protocols of DNA extraction from Escherichia coli and to report the more practical, faster, and less costly ones. Methods: E. coli , provided by Dr. Leão Sampaio University Center, was used. The methods used to extract bacterial DNA were: sodium dodecyl sulfate (SDS), salting out, cetyltrimethylammonium bromide (CTAB)/Phenol-Chloroform, and commercial kit for DNA isolation from Promega. The extracted DNA by all the methods was detected and assessed by the agarose gel electrophoresis. Results: The best result was that obtained by the SDS method, it showed the greatest advantage for bringing speed, low cost, and good concentration of the extracted material. The other methods showed low-quality DNA, probably due to the presence of large amounts of proteins in the cell wall of E. coli interfering with the quality of the samples. Conclusions: It was concluded that the SDS method can provide better DNA in terms of quality, which is preferred for the amplification by the polymerase (PCR) chain reaction. This will be helpful for diagnostic microbiologists because of the low cost and good quality of the extracted DNA.


INTRODUCTION
Based on Gram staining, the bacteria can be classified into Gram-positive and Gramnegative bacteria because of differences in the composition of their cell wall. 1 Gram-positive bacteria have a thick layer of peptidoglycan, making them more rigid; while Gram-negative bacteria have a thin layer of peptidoglycan, being more fragile and also an external membrane of lipopolysaccharide. 2 Escherichia coli (E. coli) is a Gram-negative bacterium within the family Enterobacteriaceae. It can be described as a short, mobile or immobile bacillus that lives in aerobic and anaerobic environments. It naturally inhabits the intestine and is present in most urinary tract infections (UTIs). 3 The microorganisms' isolation methods have certain limitations regarding sensitivity and specificity, time of diagnosis, and treatment. 4 Earlier the diagnosis, the greater the probability of targeted treatment, the better the prognosis, and the lower the chance of selection of microorganisms resistant to broad-spectrum antibiotics. 5 Prokaryotic cells have double-stranded and circular DNA, devoid of the nuclear membrane, proteins, and mitotic division. This genetic material has semiconservative replication, in addition, it has a plasmid as an extra chromosome, an optional structure related to the mechanism of resistance. 6 To obtain DNA, it must be intact, free of contaminants and interferents that may impair its analysis. The extraction methods consist of three steps: cell lysis and DNA solubilization, purification, and precipitation of this nucleic acid. 7 The cell wall rupture is performed with detergents such as Sodium Dodecyl Sulfate (SDS) and Cetyltrimethylammonium Bromide (CTAB). DNA separation occurs in a heterogeneous environment with phenol, which denatures proteins and the genetic material is dissolved in only one phase. 8 To remove the proteins, high salt concentrations are added (salting-out), so that they are separated from the DNA, which will be precipitated with alcohol (ethanol or isopropanol) to guarantee its purity. The analysis of DNA purity in sufficient quality and quantity can be verified by methods such as fluorimetry, spectrometry, and electrophoresis. 9 Electrophoresis is a simple technique that uses electric current to promote the separation of charged molecules, such as proteins and nucleic acids. This technique has been increasingly used in the laboratory routine contributing to the diagnosis and prognosis of several diseases. 10 Given the difficulty of microbial diagnostics, through traditional cultivation methods; the extraction of genetic material with low cost, fast and effective techniques became important to optimize techniques in the area of molecular genetics. Thus, this study aims to compare bacterial DNA extraction protocols to select a method that offers the best cost benefit and better DNA quality.

Sample preparation
Bacteria from the standard strain of E. coli, grown in Brain Heart Infusion (BHI), was transferred after a 24-hour incubation period to a 2 mL Eppendorf microtube. After transfer, the samples were centrifuged at 10000 rpm for two minutes and stored at a temperature of -20ºC for further DNA extraction.

Extraction methods
In this work, methods used are Sodium Dodecyl Sulfate (SDS), CTAB/Phenol-Chloroform, Salting-out [with and without Proteinase K enzyme (different time points and different temperatures were used to evaluate the activity of the Proteinase K)], and Wizard® Genomic DNA Purification Kit from Promega (for Isolating Genomic DNA from Gram Positive and Gram Negative Bacteria). 4

Extraction by SDS
In tubes containing the previously obtained bacterial pellet, 500 µL of lysis buffer (400mM Tris-HCl and EDTA, 50 mM, 500mM NaCl, 1% SDS) was added. The tubes were vortexed vigorously for 15 seconds and then centrifuged for 5 minutes at 10,000 rpm. After that, the supernatant was transferred to a new labeled tube and 1 mL of ice-cold concentrated ethanol was added. The tubes were gently shaken by inversion and centrifuged for 5 minutes at 10,000 rpm. The supernatant and the pellet were washed with 700 µL of ice-cold 70% ethanol followed by centrifugation for 5 minutes at 10,000 rpm. After discarding the supernatant, the tubes were placed inverted on absorbent paper to dry the pellet for approximately 30 minutes. DNA was resuspended in 50µL of ultrapure H 2 O and the sample was stored at -20 • C until further use. 11

Extraction with CTAB/Phenol-Chloroform
This method was performed according to Oliveira (2006), 12 where sterile 1.5 mL microtubes containing the bacterial pellet, as previously described, were added 250 µL of TE (10mM Tris HCl pH 7.6; 1mM EDTA) and subsequent addition of 100 µL of 5M sodium chloride (NaCl) and 100 µL of cetyltrimethylammonium bromide (CTAB) preheated to 65 • C. The suspension was stirred for ten seconds and incubated in water bath at 65 • C for ten minutes. Then, 750 µL of phenol/chloroform (1:1) were added and the mixture was shaken gently by inversion for ten seconds and then by centrifugation as described by Oliveira (2006). 12 Another wash was made with 200 µL of chloroform-isoamyl alcohol (24:1) and centrifugation. The DNA was precipitated with 1/10 volume of 3M sodium acetate and 3 times the volume of concentrated ethanol. After that, it was homogenized by inversion and incubated overnight at -20 • C. These were then centrifuged for twenty minutes at 15,000 rpm, the supernatant was discarded, and 500 µL of 70% ethanol at room temperature was added. Washing with 70% ethanol was repeated and the pellet dried at 56 • C in an oven. After ethanol was completely evaporated and the DNA solubilized in 50 µL of ultrapure water. The tubes were left for 20 minutes at room temperature and then stored at -20 • C. 13

Salting-out without Proteinase K (for 1h at 60ºC)
Two hundred microliters of TE (10mM Tris HCl pH 7.6; 1mM EDTA) plus 0.6% SDS was added to the 1.5 mL polypropylene tubes containing the E. coli and then incubated in a water bath for 1 hour at 60 • C. After incubation, 35 µL of NaCl (5M) was added, shaken manually and then centrifuged for 2 minutes at 13,000 rpm. The supernatant was transferred to a new tube and 400 µL of ice-cold absolute ethanol was added. The tubes were shaken by inversion and centrifuged for 10 minutes at 13,000 rpm. The supernatant was discarded and 1 mL of 70% ethanol was added, and the mixture was again centrifuged for 10 minutes at 13,000 rpm, discarded the supernatant, and repeated washing with 70% ethanol once more. The supernatant was discarded and the tubes were placed inverted on absorbent paper for 30 minutes until the residual ethanol evaporated. The pellet was resuspended in 50 µL of autoclaved ultrapure water and then stored at -20 • C until further use. 14

Salting-out with Proteinase K (for 1h at 60ºC)
The method was performed with the same protocol of salting-out without Proteinase K, but with the addition of 10µL of Proteinase K with TE (Tris HCl 10mM pH 7.6; EDTA 1mM) plus SDS 0.6%.

Salting-out with Proteinase K (for 2h at 42 • C)
The method was according to Abrão et al (2005) 14 with some modifications as follows. In most of the DNA extraction protocols that use the enzyme Proteinase K (PK), the incubation time is 2 hours at 42 • C, which makes the method too long. So to optimize the run time of this methodology, we used the incubation time at 60 • C for 1 hour and the 2h time at 42 • C and thus evaluate the effectiveness of the enzyme at a higher temperature and shorter time. 200µL of TE (10mM Tris HCl pH 7.6; 1mM EDTA) plus 0.6% SDS was added to 1.5 mL microtubes containing the Staphylococcus aureus bacteria and 5 µL of PK (10mg/mL). The samples were incubated in a water bath for 1 h at 60 • C and 2 h at 42 • C at separate times. After incubation, 35µL of 5M NaCl was added and subsequently, the tubes were shaken vigorously. The subsequent steps were performed as described above.

Extraction with Promega kit
One mL of the bacterial suspension, from the culture obtained as described above, was centrifuged for 2 minutes at 13,000 rpm. The supernatant was discarded after centrifugation. The pellet obtained was resuspended in 480 µL of TE buffer (10mM Tris HCl pH 7.6; 1mM EDTA) and then 600 µL of Nuclei Lysis Solution and 10 µL of PK were added and resuspended gently with a micropipette. Subsequent steps were performed according to the manufacturer's instructions. 15

Evaluation of DNA yield and its integrity
The integrity of DNA obtained was analyzed by electrophoresis in 1% agarose gel, where 8 µL of the sample was mixed in 0.5 µL of intercalante Blue Green Loading Dye I (LGC Biotechnology) and after running at 70 volts per 30 minutes and visualized in transilluminator. To evaluate the yield of the purified DNAs, the DNA Ladder 1 Kb marker (KASVI) was used.

RESULTS
The comparison between the protocols tested was performed by the amount of final DNA extracted from the E. coli samples in each protocol tested. The results can be observed in Figure 1, which presents the quantities and quality of the extracted DNA samples. The protocol that obtained the highest concentration of DNA (ng/µL) was the method SDS, which is the one of highlights of being a fast and simple method. The protocol that provided the extraction of DNA in lesser concentration was the Kit Promega (6).  Table 1 shows the results obtained after agarose gel electrophoresis 1% for quantification of the extracted DNA. For the evaluation of the DNA yield, it was compared to the KASVI DNA Ladder 1KB marker, which contains its mass in 5 ng/µL known, thus having as a base to calculate the samples according to the quantity inserted in the gel.  16 the SDS method is more efficient in terms of time and quality of the extracted DNA, besides presenting satisfactory concentration and low cost, being accessible for the development of research in several laboratories. In the study of Baratto and Megiolara (2012), 17 when comparing the DNA extraction by the SDS method of Gram-positive and Gram-negative bacteria, different results were obtained regarding the quality of the samples, suggesting that the distinct composition of the cell wall of these microorganisms may directly interfere with the test since there is a greater difficulty with Gram-positive bacteria, thus requiring more complex methodologies to obtain their genetic material.
In the present study, the protocol that provided the DNA extraction in lower concentration was using the Promega kit, not corroborating with Lipay and Bianco (2015), 18 since they report that the commercial kits for extraction of DNA present with greater frequency, better quality and purity than the manual methods, being the only disadvantage corresponding to the values of the tests.
Salting out without Proteinase K for 1 hour at 60 • C and with Proteinase k 1 hour at 60 • C and 2 hours at 42 • C have obtained similar results, although with the use of Proteinase K the test becomes more expensive, and the temperature decrease and time increase makes the test more time-consuming, being more effective, practical and fast the method Salting out without Proteinase K 1 hour at 60 • C.
The protocols with Proteinase K and CTBA are widely used and give good results. However, the use of phenol and chloroform are toxic to the environment and dangerous when handled, thus indicating their replacement by saline solution. 19 According to Bonatto and Breve (2014), 2 SDS extraction of E. coli DNA is effective in obtaining genomic DNA, but for plasmodial DNA extraction, the results are not so favorable.
The SDS is a detergent agent that solubilizes lipids, is a denaturant of proteins and also breaks non-covalent inter-subunit bonds of oligomeric proteins. It is also used to make sure that no nuclease that does not depend on Mg +2 causes any degradation. Commercially available kits, in general, result in good quality purifications for use in the most varied molecular techniques, including PCR. However, its main disadvantage is the high cost per sample. 20 The CTBA/phenol-Chloroforic test resulted in the same concentration obtained with salting out, however, is a test that presents a higher cost due to the need to use two reagents: phenol and chloroform. In addition, it has as disadvantage the risk to those who administer these substances, it is toxic to health. 19 Andreatti Filho et al. (2011) 21 verified after extracting Salmonella DNA, that the material obtained by CTAB presented a higher quality of sharpness (without the formation of nonspecific bands) than that obtained by the SDS method, and quality similar to that of the product extracted by the phenol-chloroform method, but with less intensity compared to the extraction method with guanidine thiocyanate.
Phenol causes protein denaturation efficiently. Chloroform is also used as a denaturing agent for the proteins contained in the sample. The mixture of phenol and chloroform is very efficient to deproteinize and its action is based on the hydrophobic property of proteins that have an affinity for organic solventes. 13

CONCLUSIONS
The extraction by the SDS method obtained DNA of better quality, satisfactory for polymerase chain reaction (PCR). It is a time-efficient method, that presents well quality of the extracted DNA, satisfactory concentrations, and low cost being accessible for the development of research in several laboratories. The other tested methods, presented lowquality DNA, although with good performance except with the Kit Promega, which can be explained by the presence of a large number of proteins in the E. coli cell wall, thus presenting the presence of contaminants by proteins in the electrophoresis analysis, interfering in the quality of the samples. Despite the results obtained, using different bacterial DNA extraction methodologies, it is suggested that further studies are needed. Despite the great advances in the area, still has a wide field to be explored that can generate many benefits in microbial diagnosis, with low cost and good effectiveness.