Genomic DNA Extraction from Bacteria

David A.K.E., Fernando M.F., Oliv L., and Tutor M.V. National Institute of Molecular Biology and Biotechnology University of the Philippines, Diliman, Quezon City November 27, 2013 ABSTRACT The isolation and extraction of genomic DNA is an important first step in many molecular biology applications. Yield and purity are important factors determining the quality of the resulting In this extracts, analysed with techniques such as NanoDrop and UV Spectrophotometry.

experiment, we attempt to extract genomic DNA of the bacterial strain Escherichia coli, using proteinase K and cetyltrimethyl ammonium bromide (CTAB) extraction methods. We found that proteinase K aided with CTAB successfully degraded protein components while CTAB additionally facilitates removal of residual proteins and contaminants, thereby increasing purity of genomic DNA extraction. UV spectrophotometry showed acceptable absorbance readings at 260 nm and 280 nm, with a class data set average A260/A280 ratio of 1.98 indicative of pure DNA extraction. Finally, quantification via agarose gel electrophoresis showed an almost-consistent linear band at the 23.13kbp mark across the entire class data set, further confirming bacterial genomic DNA extraction. Introduction The first step in many molecular biology applications is to isolate and extract purified genomic DNA. In general, they involve the disruption and lysis of the starting material, removal of proteins and contaminants, and finally the recovery of DNA (Moore & Dowhan, 2002). For bacterial organisms such as the gram-negative Escherichia coli (E. coli), most commonly used protocols apply some type of lysozyme/detergent lysis followed by nonspecific protease incubation and a series of phenol/chloroform/isoamyl alcohol extractions before alcohol precipitation of the nucleic acids (Wilson, 1997). Since bacteria are known to produce high levels of exopolysaccharides which may interfere with enzymatic activities in downstream applications, cetyltrimethyl ammonium bromide is introduced as a binder to both polysaccharides and residual proteins, to ensure an effective means of removing this group of contaminants. Agarose gel electrophoresis (AGE) and ultraviolet (UV) spectrophotometry are two techniques commonly used in the analysis of purified DNA. (Voytas, 2000). Standard agarose gels are an effective means of separating and identifying DNA fragments from ~0.5 to 25kb, visualized upon the illumination of UV light Meanwhile, spectrophotometry readings allow the measurement of the purity of the extracted DNA and the identification of contaminants, using absorbance ratios from A260, A280, and A230. Nucleic acids absorb maximally at 260nm, proteins at 280nm, and inorganic contaminants at 230nm (Wilfinger, 2013). In this experiment, we attempted to extract genomic DNA from the bacterial strain E. coli using the proteinase K method plus CTAB extraction with the aim of acquiring a yield with high nucleic acid

purity. Agarose gel electrophoresis and UV spectrophotometry are used to quantify and analyze DNA purification results. Materials and Methods Reagent Preparation To prepare a 24:1 solution of chloroform/isoamylalcohol, 24 ml of chloroform was mixed with 1 ml of isoamyl alcohol. To make the hexadecyltrimethyl ammonium bromide (CTAB)/sodium chloride (NaCl) solution 0.41 g of NaCl was dissolved in 8 ml deionized distilled water (ddH2O), to which 1 g of CTAB was added. The solution was adjusted to a final volume of 10 ml with ddH2O to make a final concentration of 10% CTAB in 0.7 M NaCl. A proteinase K-sodium dodecyl sulfate (SDS) solution with a concentration of 100 g/ml proteinase K in 0.5 % SDS was generated by mixing 3 l of 20 mg/ml proteinase K with 30 l of 10% SDS. To prepare a 25:24:1 solution of phenol/chloroform/isoamylalcohol, 10 ml of the chloroform/isoamylalcohol mixture was taken and mixed with 10 ml of phenol. To make 50 ml of 5 M NaCl solution, 14.61 g solid NaCl was dissolved in ddH2O and filled up to 50 ml. For the TE (hydroxymethyl aminomethane-ethylenediaminetetraacetic acid) buffer, 0.5 ml Tris was mixed with 0.1 ml EDTA and adjusted to pH 8.0, and then filled up to 50 ml with ddH2O. Cell Disruption, Lysis, and Genomic DNA Isolation An overnight bacterial culture (E. coli) was used to extract bacterial DNA. In the first step 1.5 ml of the culture was centrifuged for 2 minutes at 8000 rpm at room temperature (RT) and the supernatant was discarded. To resuspend the pellet, 567 l of Tris-EDTA buffer was added. Thirty three microliters of the proteinase K-SDS solution was added to the solution and incubated at 37C for 1 hour. After incu bation, 100 l of 5 M NaCl was added and the solution was gently mixed until a white precipitate was visible. After pipetting 80 l of the CTAB/NaCl solution, the microcentrifuge tube was heated to 65C for ten minutes. Post heating, 780 l of chloroform/isoamyl alcohol (24:1) solution was mixed and the solution was centrifuged for 5 minutes at 10,000 rpm (RT). After the aqueous supernatant was pipetted out to a fresh microcentrifuge tube, an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) solution was added and mixed by inversion. The mixture was centrifuged for 5 minutes at 10000 rpm (RT). The supernatant was transferred to a fresh microcentrifuge tube and 0.6 volume of isopropanol was added. The mixture was centrifuged for 5 min at 10000 rpm (RT) and the resulting supernatant was discarded while the pellet was washed with 500 l of 70 % ethanol. The pellet-ethanol mixture was then centrifuged for 5 minutes at 10,000 rpm (RT). Then, the supernatant was removed by decanting and aspiration while the pellet was air-dried. To resuspend the pellet, 25 L of TE buffer was added to the tube which was flicked afterwards. DNA Quantification and Analysis For UV spectrophotometry, 5 l of the extracted DNA was diluted in 495 l TE buffer and was placed in a quartz cuvette. Five hundred microliters of TE buffer in a quartz cuvette was used as a blanked sample. The spectrophotometer was adjusted to the absorption reading at 260 and 280 nm and the DNA concentration (ng/l) was determined. For NanoDrop, 1L of the extracted DNA was analyzed with ThermoScientific NanoDrop. The concentration in ng/L and the Warburg-Christian ratios of each sample was acquired.

For agarose gel electrophoresis, 0.24 g agar was dissolved in TAE buffer with heating. Ethidium bromide (EtBr) was added to a final concentration of 0.5 g/ml and the gel was mixed thoroughly. The agarose solution was poured into the mold and the gel was left to set for 30 minutes. The gel was submerged in TAE buffer and the samples were loaded. For each sample, 5 L of the extracted DNA was mixed with the 1 L gel loading buffer and the mixtures were transferred into the wells. The gel was run at 100 V until the blue dye reached 3/4 of the gel and was visualized using a UV transilluminator afterwards. Results Agarose Gel Electrophoresis Separation of purified genomic DNA extracts of E. coli was visualized using standard agarose gel electrophoresis. Figure 1 shows the group data set (wells 5-19) showing similar-patterned DNA fragments ranging from ~0.5 to 23.13kbp.

Figure 1. Agarose gel electrophoresis (0.8% agarose, 1X TAE; 100 V, 30 min) of the bacterial genomic DNA extracted using the CTAB method. Well 4 contains the Lambda-HindIII marker (Invitrogen); wells 5 to 19 contain the extracted bacterial genomic DNA (wells 6 to 7 contains the same sample). A common band in line with the 23.13 kbp marker can be observed in all of the wells except in well 14. Prominent smearing can be observed in majority of the wells in the area corresponding to molecular weights (MW) below 2.02 kbp, particularly in well 10. Well 14 has a very faint and almost undetectable profile. Curved bands are also prominent in the whole AGE profile. Spectrophotometry As can be observed in Figure 2, the samples gave out varied concentrations but the values of their purity were close to each other. The highest concentration obtained was that of sample 10 (29.20 ng/L) while the lowest was that of sample 14 (0.62 ng/L). On the other hand, the highest A260/A280 ratio obtained was that of sample 19 (2.12) while the lowest was that of sample 12 (1.73). The average concentration of the samples was 13.62 ng/L and the average A260/A280 ratio was 1.98.

Figure 2. Concentration and purity of the extracted bacterial genomic DNA. The concentrations were obtained by measuring absorbance at 260 nm using the microvolume capability of the NanoDrop 2000c UV-Vis Spectrophotometer while the purities were obtained by taking the ratio of the absorbance value of the samples at 260 nm to at 280 nm. The concentrations presented are that of a 5 L aliquot of the 25 L prepared sample solution diluted to 500 L (See Appendix A for raw data). The black bars present the concentration of each of the samples solutions while the orange bars present their corresponding purity (A260/A280). The sample identities correspond to their well numbers in the gel for the agarose gel electrophoresis in Figure 1. Discussion DNA Extraction Initial centrifugation of the bacterial culture was done to concentrate the bacteria at the bottom of the tube, allowing elimination of culture media. Tris-EDTA (TE) buffer served to maintain physiological conditions, allowing enzymes to remain active and preventing DNA degradation (Xiao, 2006). Proteinase K was used to degrade cellular proteins, while SDS served as the permeabilizing agent. Incubation was done for a long amount of time to allow SDS to break the membrane and for proteinase K to completely degrade the proteins. SDS also protects the DNA from degradation of nucleases, as well as disrupt hydrophobic interactions between nucleic acids and proteins, further isolating the nucleic acids (Wilkie, 1996). 5 M of NaCl was then added to the lysed bacteria to prevent precipitation of the nucleic acids together with cellular debris. CTAB allows a cleaner extraction as it complexes with both polysaccharides and residual protein. These complexes are then removed in the following phenol-chloroform-isoamyl alcohol extraction (Wilson, 1997). Chloroform dissolves proteins, lipids, and polysaccharides, which all gather in the organic layer, while phenol binds to proteins and denatures them (Somma, n.d.). Isoamyl alcohol prevents foaming of the solution and also aids in phase separation, while phenol dissolves proteins (Ausubel et al.). Ethanol is used to precipitate the nucleic acids out of the solution, which it accomplishes in the presence of monovalent cations such as sodium ions (Zumbo, n.d.). DNA Quantitation and Analysis Agarose gel electrophoresis (AGE) is a laboratory technique that is used to separate mixtures of biomolecules, such as DNA, RNA, or proteins by size, length, or charge (Kryndushkin et al, 2003). An electric field is applied which moves the molecules through an agarose matrix, separating them in the process (Sambrook & Russel, 2001). The AGE profiles of all the DNA extracts, except for well 14, showed a common band collinear with the 23.13 kbp mark (Figure 1). This implies that extraction of bacterial genomic DNA was successful

(Wang et al., 2013). Prominent smearing was more evident in wells 5, 7, 9, 10, 13, 17, 18, and 19 at the same mark, which may indicate that too much DNA may have been loaded onto the gel (Dube, 2001). If so, the smearing in these wells is consistent with these samples having the highest concentrations, as shown in Figure 2, since the sample amount taken from each extract was constant. The smearing at the lower half of the gel can be attributed to plasmid DNA and sheared DNA/RNA, as well as proteins left in the solution. The faint bands at the 23.13 kbp mark for wells 11, 12, 14, and 15 are congruent with the low concentrations as measured with spectrophotometry. DNA loaded into these wells may be increased to improve the resolution, but should not exceed 50 ng/band (Dube, 2001). Sample 14 was washed twice with ethanol, while Sample 11 was vortexed with a mixture of TE buffer, SDS, and Proteinase K. Excess ethanol would have decreased the binding affinity of EtBr to the DNA, giving poor resolution on the AGE profile (Li et al., 2001); also, the first ethanol wash of Sample 14 was decanted without centrifugation, which most likely lead to accidental disposal of nucleic acids. The error in the procedure for Sample 11 may have increased the DNAs susceptibility to degradation. With extended vortexing, Sample 11s extracted nucleic acids may have been mechanically sheared, resulting in the faint band at the 23.13 kbp mark. Smiling or curved bands can be observed throughout the gel, which may have been caused by improper electrophoretic conditions (Dube, 2001). Voltage may be decreased so that samples are not forced to move too fast as they are ran through the gel (Lucotte & Baneyx, 1993). Spectrophotometric methods were then employed to measure the purity of extracted genomic material. The Warburg-Christian method relates the absorbance of each biomolecular group to the amount of each kind of biomolecule contained in the sample (Warburg & Christian, 1942). With this method, one can quantify and discern the contamination present in a sample. The ratio of the absorbance values of the sample at 260 and 280 nm indicates if the samples are contaminated and the amount of contamination. For pure DNA, the 260/280 ratio is 1.8, while the 260/280 ratio for pure RNA is 2.0 (Warburg & Christian, 1942). Based on these values, majority of the extracts contain more RNA than DNA, with Sample 10 having an almost-pure RNA content. Usually, the absorbance values at 230 nm are also used to test for contamination due to inorganic compounds but the absorbance values obtained using the UV spectrophotometer are inconclusive. This is most likely due to the high dilution resulting in very low concentrations of the samples tested. Summary and Conclusions The isolation of genomic DNA from Escherichia coli was accomplished by applying extraction with proteinase K and CTAB method. DNA quantification methods such as UV spectrophotometry and AGE validated the concentration and purity of the yield, although we were not able to quantify and identify contaminants due to unreliable absorbance readings at 230 nm. The yield average of ~136mg/100ml and the extraneous bands showing in the agarose gels are indicative of likely presence of contaminants in spite of several washing cycles. But with an average A260/A280 ratio of 1.98, the extracted DNA is within the range at which it can be called pure.

Cited Literature Christian, W, and O Warburg. "Isolation and crystallization of enolase." Biochem. Z. 310:384-421 (1942): 310-384. Print. Invitrogen Quick Reference Card. "E-Gel 96 Gels." Life Technologies Corporation. N.p., 18 Apr. 2005. Web. 26 Nov. 2013. <http://tools.lifetechnologies.com/content/sfs/manuals/egel96_qrc.pdf>. Kryndushkin, D. S.. "Yeast [PSI+] Prion Aggregates Are Formed By Small Sup35 Polymers Fragmented By Hsp104." Journal of Biological Chemistry 278.49 (2003): 49636-49643. Print. WD Huang, and Y Yan. "Effect of organic solvents on DNA conformation by fluorescent probe method." PubMed - MEDLINE (5):685-7 (2001): n. pag. National Institutes of Health - Nation Library of Medicine. Web. 26 Nov. 2013. Lucotte, Gerard, and Francois Baneyx. Introduction to molecular cloning techniques. New York: VCH Publishers, 1993. Print. Moore, DM, and D Dowhan. Current Protocols in Molecular Biology. Baylor College of Medicine, Houston, Texas: John Wiley & Sons, Inc, 2002. Print. Sambrook, Joseph, and David W. Russell. Molecular cloning : a laboratory manual. 3. ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001. Print. Voytas, D. Current Protocols in Molecular Biology. Iowa State University, Ames, Iowa: John Wiley & Sons, Inc., 2000. Print. Wang, Lan. "Development of a Reference Standard of Escherichia coli DNA for Residual DNA Determination in China." PLoS ONE 8(9): e74166. (2013): n. pag. PLoS ONE. Web. 26 Nov. 2013. Wilfinger, William. "Effect of pH and Ionic Strength on the Spectrophotometric: Assessment of Nucleic Acid Purity." Biotechniques 22:474-481 (1997): n. pag. ThermoScientific. Web. 26 Nov. 2013. Wilson, K. Current Protocols in Molecular Biology. Australian Institute of Marine Science, Townsville, Australia.: John Wiley & Sons, Inc. , 1997. Print.

Appendix A. Raw Data Table 1. Raw sample concentration and purities obtained using the microvolume and cuvette capability of the spectrophotometer
Sample 5 6 9 10 11 12 13 14 15 16 17 18 19 AVE NanoConc (ng/uL) 1716.5 1509.8 1990.5 2919.5 320.1 495.1 1646.1 61.7 393.5 1547.8 1183.3 2146.3 1779.8 1362.3077 CorNanoConc 17.165 15.098 19.905 29.195 3.201 4.951 16.461 0.617 3.935 15.478 11.833 21.463 17.798 13.623077 SpecConc (ng/uL) -0.6 -0.8 -1.5 -0.5 -0.3 0.3 -0.5 -0.6 -0.4 -0.2 -1.3 -0.9 0 -0.56154 NanoPurity (A260/280) 2.1 2.04 2.05 2 1.97 1.73 2.11 1.78 1.9 2.05 2.11 1.79 2.12 1.98 SpecPurity -0.66 0.55 3.03 3.7 -16.96 -0.35 0.19 14.1 15.17 -0.49 -31.09 3.38 -0.06 -0.73

*Legend: Headers with Nano are data from the microvolume method while headers with Spec are from the cuvette method. Conc concentration; Cor corrected.

B. Sample Calculations ( )( )

( )(

C. Supplementary Figure

Figure A. Lambda-HindIII marker (Invitrogen). Source: http://www.chromatrin.com/xcart/skin1/images/customer_images/lambda.gif