Newman_et._al_2010 Purification of Genomic DNA Extracted From Environmental Sources for Use in a PCR

Purification of Genomic DNA Extracted from Environmental Sources for Use in a Polymerase Chain Reaction Molli M. Newman, Jack W. Feminella, and Mark R. Liles 1 Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA INTRODUCTION The ability to amplify genomic DNA in a polymerase chain reaction (PCR) is dependent upon the purity of the DNA template. Environmental genomic DNA often contains contaminants (e.g., polyphe- nols, humic acids, polysaccharides)
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  Purification of Genomic DNA Extracted from EnvironmentalSources for Use in a Polymerase Chain Reaction Molli M. Newman, Jack W. Feminella, and Mark R. Liles 1 Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA INTRODUCTION The ability to amplify genomic DNA in a polymerase chain reaction (PCR) is dependent upon thepurity of the DNA template. Environmental genomic DNA often contains contaminants (e.g., polyphe-nols, humic acids, polysaccharides) that reduce template purity and can be difficult to remove,thereby inhibiting PCR amplification. There is thus a need for a method to purify extracted genomicDNA without reducing DNA concentration. In this protocol, extracted genomic DNA is embedded inagarose plugs and incubated in a formamide and salt (NaCl) solution to remove contaminants. TheNaCl works to deproteinize and stabilize the DNA. The formamide serves to denature the DNA (whichwill subsequently be renatured within the agarose plug) and any contaminants that may be bound tothe DNA. The purified DNA is extracted from the agarose plug using a standard commercial agaroseextraction method, and the DNA may then be used as a template for PCR. Genomic DNA purifiedusing this method has been shown to serve as an efficient template for PCR, without significant lossof DNA yield. An additional advantage of the method is that it allows the simultaneous processing of large numbers of samples at once. RELATED INFORMATION The increase in PCR efficiency using genomic DNA purified by this method is shown in Figure 1. A quantitative PCR was performed using a genomic DNA template purified by this method and theresults were compared to those from templates prepared using a commercial kit and other treatmentcombinations. Liles et al. (2008) have described a method for purifying high-molecular-weight DNA for use in the construction of metagenomic libraries (see also  Isolation and Cloning of High-Molecular-Weight Metagenomic DNA from Soil Microorganisms  [Liles et al. 2009]). However, theprotocol reported here is more rapid and is specifically designed for the purification of genomic DNAto be used as a template for PCR amplification. © 2010 Cold Spring Harbor Laboratory Press  1  Vol. 2010, Issue 2, February 1 Corresponding author ( Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5383 Protocol MATERIALS CAUTIONS AND RECIPES:  Please see Appendices for appropriate handling of materials marked with  <!> , andrecipes for reagents marked with  <R> . Reagents  Agarose (molecular biology grade; Fisher Scientific)Environmental sample of interest  2  Cold Spring Harbor Protocols METHOD Generation of Agarose Plugs and Overnight Incubation 1.  Extract genomic DNA from an environmental sample using a direct extraction method or acommercial kit. To determine the amount of environmental sample to be processed, follow published methods or the manufac-turer’s instructions. Typically, commercial kits suggest using <500 mg of environmental sample for extraction.Note that, even though commercial kits may advertise that “PCR-ready” DNA will be extracted, it is not unusual to obtain samples that cannot be readily used as a PCR template. If, after serial dilution of the DNA template,no PCR product is obtained and the positive controls have been successful, the protocol outlined below may be useful for purifying truly PCR-ready DNA. <!> Formamide (deionized, 80% [v/v] in 1.3 M NaCl) Store at 4°C. Gel extraction kit (e.g., QIAquick Gel Extraction Kit from QIAGEN or Wizard SV Gel and PCRClean-Up System from Promega)NaCl (1.3 M)Reagents for extracting genomic DNA from environmental sample (see Step 1) <R> TAE buffer (50X stock) Dilute to 1X before use. Equipment Beaker (sterile, glass, for preparing soft agarose)Equipment for extracting genomic DNA from environmental sample (see Step 1)Microcentrifuge tubes (2-mL)Micropipettors (p200 and p1000) and tips (200-µL and 1-mL)Microwave ovenTube rack Waterbath preset to 15°C FIGURE 1.  Bar graph representing amplified environmental genomic DNA isolated from red maple (  Acer rubrum ) leaf litter. Genomic DNA was purified using several treatment combinations and gel quantified prior to amplification. A stan-dardized amount of DNA (20 ng) was then used in PCR reactions (domain Bacteria primer set 338F and 518R [Øvreåset al. 1997]) containing a fluorescent probe (SYBR Green; Bio-Rad) that binds to double-stranded DNA, and the fluo-rescence was measured over time. Final DNA concentration was then calculated by comparing fluorescence of a PCRstandard of known DNA concentration to the fluorescence of each sample. All points represent standardized DNA quan-tity ± standard error. Letters indicate Tukey’s multiple comparison groupings.  2.  Prepare an appropriate volume of 2% agarose solution in 1X TAE in a sterile glass beaker. Heatin a microwave oven and let the solution cool to 45°C. Mix briefly to ensure that the agarosesolution is homogenous. 3.  Add equal volumes of 2% agarose solution and DNA extract to a 2-mL microcentrifuge tube.Mix by pipetting up and down briefly using a 1-mL pipette tip. Typical volumes would be 100 µL of 2% agarose mixed with 100 µL of DNA extract. The purpose of embedding the DNA sample in agarose is to allow rapid DNA purification and subsequent washing without the need for DNA precipitation. The agarose provides a matrix for the DNA once it is denatured by the formamide/NaCl solution, thereby allowing contaminants to move into the surrounding liquid. 4.  Allow the agarose plug to solidify in the microcentrifuge tube at room temperature. This should take <10 min. 5.  Add 5X volume of a solution of 80% formamide in 1.3M NaCl to the agarose plug. Mix by slowlyinverting the tube in a rack. Formamide denatures the DNA and NaCl stabilizes it during the incubation. If the total volume of the agarose plug is 200 µL, add 1 mL of formamide/NaCl solution.See Troubleshooting. 6.  Incubate the samples for 1 h at 15°C. Washing of Agarose Plugs and Extraction of DNA from Agarose 7.  Remove the formamide/NaCl solution. 8.  Wash the agarose plug five times with 1 mL of 1X TAE. 9.  Extract the DNA from the agarose using a commercial gel extraction kit. The DNA can now be used for PCR or it can be stored at −20°C or −85°C.See Troubleshooting. TROUBLESHOOTING Problem:  The agarose plug remains stuck in the bottom of the microcentrifuge tube. [Step 5]Solution:  It may be necessary to release the agarose plug from the bottom of the microcentrifugetube by pressing the pipette tip down the side of the agarose plug. This step will ensure totalsuspension of the agarose plug in the formamide/NaCl solution. Use of a 2-mL microcentrifugetube during washing steps may prevent this problem. Problem:  The DNA is not sufficiently pure. [Step 9]Solution:  Consider the following: 1.  Depending upon the degree of contamination, the incubation time in Step 6 may be extended toovernight at 15°C. 2.  With highly contaminated samples (i.e., those that change color owing to phenolic or humiccontaminants), it may be advisable to remove the formamide/NaCl solution during the incubationand replace it with fresh solution. Problem:  The DNA yield is insufficient. [Step 9]Solution:  Consider the following: 1.  During the DNA extraction in Step 9, make sure to follow the manufacturer’s recommendations inrecovering DNA from the agarose gel. 2.  To maximize the yield of DNA recovered from the column, try reducing the volume of the elutionbuffer (to ~30 µL) and passing the elution buffer over the column two or more times. The onlysignificant loss of DNA during the protocol occurs at this step.  3  Cold Spring Harbor Protocols  Holben WE, Jansson JK, Chelm BK, Tiedje JM. 1988. DNA probemethod for the detection of specific microorganisms in the soilbacterial community.  Appl Envirnon Microbiol   54:  703–711.Knaebel DB, Crawford RL. 1995. Extraction and purification of micro-bial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by mul-tiplex polymerase chain reaction and Southern analysis.  Mol Ecol  4:  579–591.Liles MR, Williamson LL, Rodbumrer J, Torsvik V, Goodman RM,Handelsman J. 2008. Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms.  Appl EnvironMicrobiol   74:  3302–3305.Liles MR, Williamson LL, Rodbumrer J, Torsvik V, Parsley LC, GoodmanRM, Handelsman J. 2009. Isolation and cloning of high-molecular-weight metagenomic DNA from soil microorganisms.  Cold Spring Harb Protoc   doi: 10.1101/pdb.prot5271.Ogram A, Sayler GS, Barkay T. 1987. The extraction and purificationof microbial DNA from sediments.  J Microbiol Methods   7:  57–66.Øvreås L, Forney L, Daae FL, Torsvik V. 1997. Distribution of bacteri-oplankton in meromictic Lake Sælenvannet, as determined bydenaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.  Appl Environ Microbiol   63: 3367–3373.Robe P, Nalin R, Capellano C, Vogel TM, Simonet P. 2003. Extractionof DNA from soil.  Eur J Soil Biol   39:  183–190.Roose-Amsaleg CL, Garnier-Sillam E, Harry M. 2001. Extraction andpurification of microbial DNA from soil and sediment samples. Appl Soil Ecol   18:  47–60.Steffan RJ, Goksøyr J, Bej AK, Atlas RM. 1988. Recovery of DNA fromsoils and sediments.  Appl Environ Microbiol   54:  2908–2915.  4  Cold Spring Harbor Protocols DISCUSSION Several methods exist for the purification of environmental DNA, including those that use phenol-chloroform, hexadecyltrimethylammonium bromide (CTAB), polyvinylpolypyrrolidone (PVPP), cesiumchloride density centrifugation, and hydroxyapatite column chromatographic purification (for reviews,see Roose-Amsaleg et al. 2001; Robe et al. 2003; see also Ogram et al. 1987; Holben et al. 1988;Knaebel and Crawford 1995). Steffan et al. (1988) have demonstrated that many of these methods(such as those using PVPP, cesium chloride, and hydroxyapatite) lower DNA yield. Often, a combina-tion of two or more purification methods (such as a phenol-chloroform extraction followed by use of CTAB) is required to attain adequate purification of environmental DNA. However, using numerouspurification steps not only can decrease DNA yield, but also increases sample-processing time. Thelatter is particularly disadvantageous when working with large numbers of samples. The protocoldescribed here allows multiple samples to be processed at once.The purification and amplification of environmental DNA can often be difficult because of low yields and co-isolation of contaminants. Incubation of genomic DNA in agarose plugs during for-mamide and salt treatment allows removal of contaminants without significant loss of DNA. Theprotocol described here has been shown to be effective in purifying DNA from various environmentalsources, such as soils, leaf litter, and marine corals, which have never successfully provided templates for PCR amplification. Commercial kits for genomic DNA extraction typically shear the DNA, resultingin fragment sizes <20 kb. The DNA yield obtained using this procedure will vary greatly dependingon the initial DNA concentration. After following this procedure, each of the purified DNAs yieldedabundant amplicons using ~20 ng of purified DNA as a template for PCR. REFERENCES
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