Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures

Correspondence: Konstantinos Minas, Institute for Innovation Design and Sustainability, Robert Gordon University, Saint Andrew Street, Aberdeen AB25 1HG, UK. Tel.: +44 01224 262847; fax: +44 01224 263000; e-mail: k.minas@rgu.ac.uk

FEMS Microbiology Letters, Volume 325, Issue 2, December 2011, Pages 162–169, https://doi.org/10.1111/j.1574-6968.2011.02424.x

Cite

Konstantinos Minas, Neil R. McEwan, Charles Jamie Newbold, Karen P. Scott, Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures, FEMS Microbiology Letters, Volume 325, Issue 2, December 2011, Pages 162–169, https://doi.org/10.1111/j.1574-6968.2011.02424.x

Abstract

The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.

Introduction

In recent years, the use of molecular methods such as T-RFLP and qPCR has become increasingly widespread because of their sensitivity, specificity and reliability. These molecular tools are routinely used in laboratories for the detection of single genes/organisms or for the profile analysis of complex biological systems. As a result, biases related to PCR also need to be considered (Peano et al., 2004; Bustin et al., 2009; Sipos et al., 2009). One of the most important factors in a PCR-based experiment is the quality and quantity of the template DNA, as the presence of various inhibitors in template DNA has differential effects on the outcome of a PCR amplification (Wilson, 1997; Huggett et al., 2008).

The wide applicability of PCR-based techniques has rendered these methods paramount in scientific research (Hubner et al., 2001; Pierson et al., 2003; Burns et al., 2004). Cross-laboratory data comparison requires standardization, and this has been addressed by the establishment of minimum information guidelines. In the particular case of qPCR, the Minimum Information for Publication of Quantitative Real-Time PCR experiments (MIQE) has become available (Bustin et al., 2009). Moreover, the Minimum Reporting Guidelines for Biological and Biomedical Investigations (MIBBI Project) was developed to facilitate further coordination in research (Taylor et al., 2008). Selection of an appropriate DNA extraction and purification protocol is essential for most downstream applications in molecular biology. To date, an array of chemical, mechanical and enzymatic methods have been developed for the extraction and purification of DNA from a variety of samples (Tsai & Olson, 1991; Wilson et al., 1991; Roman & Brown, 1992; Corbisier et al., 2007). The physical and chemical properties of nucleic acids are quite similar to those of some commonly co-precipitated PCR inhibitors. As such, most DNA extraction and purification methods are characterized by inherent biases that are manifested in the later steps of PCR amplification. (Rossen et al., 1992; Miller et al., 1999). In the particular cases of PCR amplification of samples originating from plant cells, food, gut bacteria or soil, the presence of PCR inhibitors can be particularly high in the starting material (Demeke & Adams, 1992; Bickley et al., 1996; Monteiro et al., 1997; Watson & Blackwell, 2000) because of the co-precipitation of contaminants such as humic acids and phenolic substances. These substances have adverse effects on PCR amplification and thus can affect the generation of accurate and reproducible experimental data.

In this manuscript, we describe the optimization of a DNA extraction method, developed for DNA extraction from rumen fluid and solid plant material, but that is equally useful for a variety of samples. DNA purified using this method was compared with DNA extracted using previously published manual methods and commercially available kits. The yield and quality of the extracted DNA was assessed by UV spectrophotometry. In all cases, the CTAB method of DNA extraction produced high yields and consistent quality of DNA. Moreover, the DNA templates obtained using the finalized protocol could be used successfully as templates in qPCR amplification, therefore confirming the lack of PCR inhibitors in these samples.

Materials and methods

Plant materials

Seeds originated from three genetically modified plants were ground to 1 mm 3 prior to being used for DNA extraction. The plant isotypes used were as follows: InVigor 5020 Argentine canola (Bayer, Germany), Herculex I maize (DOW Agrosciences LLC and Pioneer Hi-Bred International Inc., Canada) and RoundupReady soya (Monsanto, Canada).

Rumen fluid acquisition

Rumen fluid used in the standard protocol was harvested from a ruminally cannulated sheep. The rumen fluid used in the high-throughput method was pooled from four ruminally cannulated cows. Prior to use, rumen fluid was strained in four layers of cheesecloth at 39 °C under continuous flow of CO₂.

Bacterial cultures

Pure cultures of Escherichia coli (Gram-negative) and Enterococcus faecalis (Gram-positive) were grown to stationary phase prior to DNA extraction from fresh cultures. Escherichia coli cultures were incubated at 37 °C overnight in selective LB (Oxoid, Cambridge, UK) containing 5 μg mL −1 chloramphenicol (Fisher, Leicestershire, UK). Precipitation of cells was avoided by shaking bacterial cultures at 250 r.p.m. on a rotating platform. Enterococcus faecalis was cultured without shaking for 48 h at 39 °C in nonselective M2GSC media (Miyazaki et al., 1997) under anaerobic conditions.

DNA extraction methods (commercial kits)

Ten millligrams of ground seed mixed with 10 μL of rumen fluid was tested as the starting material for DNA extractions using the Wizard SV Genomic DNA purification kit (Promega, Southampton, UK) and the DNeasy Plant Mini Kit (Qiagen, West Sussex, UK). 20 mg of lyophilized rumen fluid: ground plant seed material was used for the DNA extractions using the QIAamp DNA stool Mini kit (Qiagen). DNA extractions using commercial kits were performed according to respective manufacturers’ instructions. In addition, three previously published methods for DNA extraction from plants were also tested [Kang & Yang, 2004; Alexander et al., 2006, Community Reference Laboratory, (CRLV04/05XP)]. In these instances, the recommended amount of starting material was used for each extraction.

DNA extraction methods (manual)

Three protocols for DNA extraction using the CTAB-based DNA extraction method are described, with the method of choice dependent on the extraction scale required. The CTAB lysis buffer contained 2% w/v CTAB (Sigma-Aldrich, Poole, UK), 100 mM Tris–HCl (pH = 8.0; Fisher), 20 mM EDTA (pH = 8.0; Fisher) and 1.4 M NaCl (Fisher). The pH of the lysis buffer was adjusted to 5.0 prior to sterilization by autoclaving (Doyle & Doyle, 1987).

1. Standard method in 2.0-mL microcentrifuge tubes: The original samples used in all the protocols described herein consisted of 1.8 mL of rumen fluid and 50 mg of ground plant seed material. Samples were lyophilized at − 40 °C for 48 h and bead-beaten on a prechilled rack at − 80 °C for 1 min using 8-mm glass beads (Fisher). For the optimized protocol, 50 mg of lyophilized material was thoroughly mixed with 900 μL of CTAB lysis buffer. All samples were incubated at 65 °C for 60 min before being centrifuged at 12 000 g for 5 min at 4 °C. Supernatants were transferred to fresh 2-mL microcentrifuge tubes and 900 μL of phenol: chloroform: isoamyl alcohol (25 : 24 : 1, pH = 6.7; Sigma-Aldrich) added for each extraction. Samples were mixed thoroughly prior to being incubated at room temperature for 10 min. Phase separation occurred by centrifugation at 12 000 g for 15 min at 4 °C, and the upper aqueous phase was re-extracted with a further 900 μL of phenol:chloroform:isoamyl alcohol. Next, samples were centrifuged at 12 000 g for 10 min at 4 °C, and the upper aqueous phases were transferred to fresh 2-mL microcentrifuge tubes. The final extraction was performed with 900 μL of chloroform: isoamyl alcohol (24 : 1), and layer separation occurred by centrifugation at 12 000 g for 15 min at 4 °C. Precipitation of DNA was achieved by adding the upper phase from the last extraction step to 450 μL of isopropanol (Sigma-Aldrich) containing 50 μL of 7.5 M ammonium acetate (Fisher). Samples were incubated at −20 °C overnight, although shorter incubations (1 h) produced lower DNA yields. Samples were centrifuged at 7500 g for 10 min at 4 °C, and supernatants were discarded. Finally, DNA pellets were washed three times in 1 mL of 70% (v/v) ethanol (Fisher). The final pellet was air-dried and re-suspended in 200 μL of 75 mM TE buffer (pH = 8.0; Sigma-Aldrich).
2. High-Throughput (96-well plate format): Twenty millligrams of starting material was used for each DNA extraction in the high-throughput format. 110 μL of CTAB lysis buffer was added to each sample, and samples were incubated at 65 °C for 60 min. Samples were extracted twice with 110 μL of phenol: chloroform: isoamyl alcohol (25 : 24 : 1, pH = 6.7; Sigma-Aldrich) and once with 110 μL of chloroform: isoamyl alcohol (24 : 1; Sigma-Aldrich). After each extraction, samples were centrifuged at 3000 g for 60 min at 4 °C, and supernatants were discarded. Supernatants were transferred in wells containing 90 μL of isopropanol (Sigma-Aldrich) and 10 μL of 7.5 mM ammonium acetate (Fisher). DNA was precipitated at −20 °C overnight, followed by centrifugation of samples at 3000 g at 4 °C for 60 min. Three ethanol washes were performed by adding 110 μL of 70% (v/v) ethanol (Sigma-Aldrich) to each sample and centrifuging for 30 min at 3000 g at 4 °C. Supernatants were discarded after each ethanol wash. Excess ethanol was removed by centrifuging the plates upside down at 300 g for 10 s at 4.0 °C. DNA pellets were air-dried prior to being re-suspended in 50 μL of 75 mM TE buffer (pH = 8.0; Sigma-Aldrich).
3. Large-scale (50-mL Falcon format): Firstly, cells were harvested in 50-mL Falcon tubes by centrifugation at 4000 g for 10 min. Growth media were discarded, and each bacterial pellet was re-suspended in 5 mL of CTAB lysis buffer. Cell lysis was achieved by incubating samples at 65 °C for 60 min. DNA was then extracted twice using an equal volume (5 mL) of chloroform: isoamyl alcohol (24 : 1; Sigma-Aldrich) each time. Cellular fractions were separated by centrifuging samples at 8000 g for 15 min, and the process was repeated. DNA was precipitated at −20 °C overnight in 5 mL of isopropanol: 7.5 M ammonium acetate (9 : 1; Sigma-Aldrich). DNA was harvested by centrifugation at 8000 g for 15 min. Finally, DNA pellets were washed twice in 5 mL of 70% (v/v) ethanol (Sigma-Aldrich), and samples were collected by centrifugation at 8000 g for 10 min. Each resultant DNA pellet was re-suspended in 5 mL of 75 mM TE buffer (pH = 8.0; Sigma-Aldrich).

DNA quantification

The quality and quantity of the extracted DNA was tested by UV spectrophotometric analysis at 260 nm using a Nanodrop ND-1000. Similarly, quantitative analysis was performed at 280 and 230 nm. Statistical significance of our data was assessed by anova . Qualitative analysis was continued by loading 10 μL of each DNA sample on a 0.8% agarose gel and performing electrophoresis at a constant current of 70 V for 90 min.

qPCR Analysis

The lack of PCR inhibitors in the DNA templates was verified when the purified DNA was used in qPCR applications, using the Biorad iQ5 system. Here, the extracted DNA samples were used in qPCR amplifications for transgenic and endogenous plant genes as well as for the detection of bacterial 16S rDNA. The sequences of the primers used in this study can be found in Table 1, and all were used at a final concentration of 0.1 μM. Template DNA was diluted to a final concentration of 10 μg μL −1 using 5 μg mL −1 of herring sperm DNA (Promega) as a diluent. One microlitre of template was added to each reaction, and the qPCR amplifications were performed in 15-μL reaction volumes using the SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich) according to the manufacturer's instructions. The amplification conditions for the qPCR amplification of Lectin were as follows: initial denaturation at 95 °C for 10 min followed by 43 cycles of incubation at 94 °C for 30 s, annealing at 59 °C for 30 s and incubation at 72 °C for 30 s. Amplification of invertase was performed by 50 cycles of incubation at 94 °C for 30 s followed by a 45-s incubation at 63.2 °C. After the initial denaturation, amplification of cruciferin was performed at 40 cycles of incubation at 95 °C for 30 s, 59 °C for 1 min and 72 °C for 30 s. Finally, for the qPCR amplification of 16S rDNA, the initial denaturation at 95 °C for 5 min was followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s and elongation at 72 °C for 30 s. In the particular case of 16S rDNA amplification, a final elongation at 72 °C for 10 min was also included. In all cases, melting curve analysis was performed at a temperature range of 65–95.1 °C.

Sequences of qPCR primers used in this study. The table below contains the primer sequences used in this study, as well as their specificity and the expected amplicon sizes

Sequences of qPCR primers used in this study. The table below contains the primer sequences used in this study, as well as their specificity and the expected amplicon sizes

Results and discussion

Qualitative and quantitative analysis of the DNA obtained during the optimization of the DNA extraction method was performed by UV spectrophotometry (Table 2; Supporting Information, Tables S1–S4). Although starting quantities of rumen fluid and plant material differ because of limitations associated with the different techniques, clearly the yields of DNA obtained were extremely variable, ranging from undetectable to 800 ng μL −1 . Generally, the highest yields combined with the optimal A_{260 nm}/A_{280 nm} ratios were obtained using CTAB. The statistical significance of the data obtained by this method was analysed by anova (Table 3). This analysis demonstrated that the reagents used for the extraction had significant effects on the yield of DNA extracted from rapeseed and maize. Namely, the yield was significantly higher when DNA was extracted twice with phenol : chloroform : isoamyl alcohol, than when phenol was omitted from the extraction reagent. Inclusion of phenol in the extraction buffer did not yield higher amounts of DNA for soya, but the quality of DNA was significantly higher when the extraction reagent included phenol. The amount of starting material used for each extraction did not have any significant effects for rapeseed. On the other hand, 50 mg of starting material appeared to be the optimum for DNA extracted from maize. In the particular case of soya, the amount of extracted DNA appeared to be directly correlated with the amount of starting material used for the extraction (Table 3).

UV spectrophotometric analysis of samples extracted with the different methods described in this manuscript. Starting material used in each method, as well as the resulting purity and yield of the product DNA

UV spectrophotometric analysis of samples extracted with the different methods described in this manuscript. Starting material used in each method, as well as the resulting purity and yield of the product DNA

Statistical significance of factors influencing DNA yield and/or quality in the CTAB-based protocol. The P-values described below were calculated by anova . The inclusion of phenol in the DNA purification step consistently produced high yields of DNA, whereas the amount of starting material used for each extraction did not have such an effect

P, Phenol; C, Chloroform; IAA, Isoamyl alcohol. Statistically significant results are shown in bold.

Statistical significance of factors influencing DNA yield and/or quality in the CTAB-based protocol. The P-values described below were calculated by anova . The inclusion of phenol in the DNA purification step consistently produced high yields of DNA, whereas the amount of starting material used for each extraction did not have such an effect

P, Phenol; C, Chloroform; IAA, Isoamyl alcohol. Statistically significant results are shown in bold.

Agarose gel electrophoresis verified the results obtained by UV spectrophotometry. Thus, exclusion of phenol from the extraction buffer resulted in the presence of contaminating substances in soya and rapeseed DNA that were retained in the wells (Fig. 1). As these substances did not appear to have any significant effects on the A_{260 nm}/A_{280 nm} ratio obtained by the Nanodrop, it was assumed that the co-precipitating substances were humic acids. Humic acids absorb UV light at a similar wavelength to that of nucleic acids (254 nm), thus would not affect the A_{260 nm}/A_{280 nm} ratio, but they are unable to penetrate agarose gels. In contrast, humic acids are extremely inhibitory for PCR amplifications (Lead et al., 2003; Weishaar et al., 2003); thus, the inclusion of phenol in the extraction buffer was considered essential for the isolation of high-purity DNA templates.

Agarose gel electrophoresis of the genomic DNA obtained from the optimisation of the CTAB extraction method. DNA samples contained in this figure were obtained from rumen fluid containing RoundupReady soya (a), Herculex I maize (b) or InVigor 5020 Oilseed Rape (c). Qualitative assessment of the genomic DNA demonstrated that the inclusion of Phenol in the extraction buffer significantly increased the yield of DNA (lanes 6–9, 14–18 and 23–26). More significantly, the purity of extracted DNA was significantly lower for samples where phenol was not used in the extraction. This was also manifested by the presence of impurities in lanes 2–5, 10–13 and 19–22, demonstrated by substances remaining in the wells. The amount of ground plant material used in each extraction, also appeared to affect the yield but not the purity of the DNA extracted. The increasing intensity of smears appeared to correlate with the increasing amount of ground plant material used in for the extraction of DNA. More information on the samples present in this gel can be found on Supporting Information, Tables S1–S4.

The quality of extracted DNA was finally confirmed by using selected samples as templates in qPCR amplifications. The extraction of plant DNA was verified by the qPCR amplification of respective plant housekeeping genes, while the successful co-extraction of bacterial genomic DNA was verified by qPCR amplification of 16S rDNA (Table 4). As the amount of DNA included in each qPCR amplification had been standardized at 5 μg mL −1 , the presence or absence of PCR inhibitors could be assessed by comparing the threshold cycle (C_t) of each reaction, which increased in proportion to the amount of inhibitors present. Statistical significance of the qPCR amplification data was performed in anova (Table 4). Amplification of plant and bacterial genes did not appear to be significantly influenced by the amount of starting material used in the extractions. The only exception to this observation was the amplification of cruciferin in rapeseed. In this instance, extraction of DNA from 50 mg of plant material appeared to be optimum, as manifested by earlier detection of the cruciferin qPCR amplicon.

Statistical significance of factors influencing qPCR amplification of housekeeping genes of bacterial and plant origin. The table shows the average C_t values obtained after qPCR amplification of respective genes. The P-values described below were calculated by anova

Statistically significant results are shown in bold.

Statistical significance of factors influencing qPCR amplification of housekeeping genes of bacterial and plant origin. The table shows the average C_t values obtained after qPCR amplification of respective genes. The P-values described below were calculated by anova

Statistically significant results are shown in bold.

The effects of Proteinase K and/or RNase H inclusion in the lysis buffer were examined because historically, these proteins have been used in DNA extractions for improved yield and quality of the extracted DNA. Namely, proteinase K is a serine protease that catabolizes a broad spectrum of proteins, including nucleases (Gross-Bellard et al., 1973; Kasche et al., 1981). RNase H on the other hand, specifically removes RNA from RNA:DNA complexes, therefore alleviating nuclear RNA contamination (Berkower et al., 1973). Both these proteins were subsequently removed by phenol extraction following cell lysis (Burrell, 1993). Our analysis showed that the inclusion of neither of these proteins had significant effects on the quality of extracted DNA, as manifested by the C_t in subsequent qPCR amplification of samples (Table 4).

qPCR detection of bacterial DNA was not possible when the template DNA was extracted using the Wizard SV Genomic DNA purification kit (Promega) or the DNeasy Plant Mini Kit (Qiagen). Additionally, qPCR amplification of endogenous plant genes was unsuccessful when the DNA was extracted by using the QIAamp DNA stool Mini kit (Qiagen) (data not shown). On the other hand, DNA extraction using the CTAB protocol enabled the detection of both plant and bacterial DNA in the same sample. Collectively, these data demonstrate that extraction using the CTAB protocol produces DNA of sufficient quantity and quality for use in qPCR amplification. Moreover, when compared to the commercially available kits, DNA extraction using the CTAB protocol was more cost efficient, without consistently being the most time-efficient method.

Editor: Jeff Cole

Acknowledgements

The authors would like to thank Dr Eva Morales, Mrs Nest McKain, Mrs Hilary Worgan, Mr Gary Easton and Dr Susan Greenwood for invaluable contribution in sample preparation. In addition, Dr Grietje Holtrop (Biomathematics and Statistics Scotland) provided valuable input in the statistical analysis of data. The work described in this manuscript was supported by a grant received from the Food Standards Agency (FSA; G03031). The Rowett Institute of Nutrition and Health receives support from the Scottish Government (Rural and Environment Science and Analytical Services; RESAS).