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ISSN: 2155-9821
Journal of Bioprocessing & Biotechniques

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The Determination of Factors involved in Column-Based Nucleic Acid Extraction and Purification

Jun-Jie Poh1,3 and Samuel Ken-En Gan1,2,3*
1 Bioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), 138671, Singapore
2 p53 Laboratory, Agency for Science, Technology, and Research (A*STAR), 138648, Singapore
3 Quintech Life Sciences Pte Ltd, 619933, Singapore
Corresponding Author : Samuel Ken-En Gan
Bioinformatics Institute
Agency for Science, Technology and Research (A*STAR)
30 Biopolis Street, #07-01 Matrix
138671, Singapore
Tel: 65-6478-8417, 65-6407-0584
Fax: 65-6478-9047
E-mail: [email protected]
Received April 25, 2014; Accepted June 06, 2014; Published June 12, 2014
Citation: Poh JJ, Gan SKE (2014) The Determination of Factors Involved in Column-Based Nucleic Acid Extraction and Purification. J Bioprocess Biotech 4:157 doi: 10.4172/2155-9821.1000157
Copyright: © 2014 Poh JJ et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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DNA extraction methods such as plasmid minipreps, gel, and PCR purifications, are indispensable techniques for genetic manipulations. There are numerous factors that contribute to the efficiency of these processes, which determine the success of complex downstream molecular analytics and diagnostic tests. To study and optimize these factors, we compared our own proprietary buffers to commercially available column-based kits, utilizing their spin columns and protocols. Through systematic substitution of the buffers in the kits with our own proprietary buffers, we selected the highest DNA yielding buffer recipes. Further analysis of the differences between the buffers showed that high concentrations and presence of certain chaotropic agents and cations are necessary for good plasmid miniprep, gel extraction, and PCR purification kits.

DNA extraction; Gel extraction; PCR purification; Column-based purifications
E. coli: Escherichia coli; SDS: Sodium Dodecyl Sulfate; TE: Tris-EDTA; OPT: Optimized; HM: Home-Made
The extraction and purification of nucleic acids are commonly used techniques to isolate genetic material from tissues, bacteria, plants, and viruses for important analytical, diagnostic and preparative downstream processes. Amongst these methods, plasmid DNA extraction was the first to be reported [1] using the tedious alkaline extraction protocol. This involved lysozyme treatment to weaken the Escherichia coli (E.coli) cell wall prior to cell lysis and selective denaturation of genomic DNA using sodium dodecyl sulfate (SDS) and sodium hydroxide. Sodium acetate is then used to neutralize the alkaline pH, resulting in the formation of an insoluble network of denatured genomic DNA, protein-SDS complexes and high molecular weight RNA. These complexes were then removed by high speed centrifugation, leaving the desired plasmid DNA in the supernatant [1].
As the protocol was labour-intensive, efforts to simplify the extraction methods gave rise to the development of the "Guanidinium Thiocynate - Phenol - Chloroform" method [2] to separate the various biomolecules through multiple liquid phases [3]. Further developments resulted in doing away with the use of hazardous chemicals (phenol and chloroform) through the use of spin columns for rapid extraction of high purity nucleic acids. Despite simplifying the process through the immobilization of plasmid DNA to the solid phase matrix (i.e. silica), plasmid extraction is still underlined by the need to disrupt bacterial cell walls, denaturation of nucleic acid binding proteins, inactivation of nucleases such as RNases, washing away of undesired contaminants, and elution of desired plasmid DNA.
At the crux, the silica solid phase matrix determines the resultant product purity and yield. For optimal DNA binding, equilibration of these silica columns by Na+ is required to break hydrogen bonds for the formation of salt bridges, allowing for spatial interaction with the negatively charged DNA. The silica membrane is then washed with ethanol to remove salts and other contaminants prior to elution of the bound DNA using low ionic strength (pH ≥ 7) buffers [3].
Based on the same principle for plasmid DNA extraction, spin columns had also been used for gel extractions and polymerase chain reaction (PCR) purifications. While these developments have contributed greatly to biomedical research, major developments of these kits are generally largely focussed on membrane material science. To complement this, we have decided to investigate whether the manipulation of chemicals in associated buffers will increase the yields that would enable researchers to tweak their existing commercial kits for improved yields.
Materials and Methods
Investigation of miniprep buffers
Proprietary buffers: Proprietary equilibration buffers (P-BK1 and P-BK2); resuspension buffers (P-P1); lysis buffers (P-P2); neutralization buffers (P-P3-1 and P-P3-2); binding buffer (P-W1); wash buffers (PW2- 1 and P-W2-2) and elution buffers (P-EB1, P-EB2, and P-EB3) for plasmid extraction with the following ingredients were prepared:
P-BK1 ----- NaCl, MOPS
P-BK2 ----- NaOH (> 1M [Na+] than P-BK1)
P-P1 ----- Tris Base, EDTA, RNase A
P-P2 ----- SDS, NaOH
P-P3-1 ----- C2H3KO2
P-P3-2 ----- NH2C(=NH)NH2 • HCl , C2H3KO2 ( pH< P-P3-1)
P-W1 ----- NH2C(=NH)NH2 • HCl
P-W2-1 ----- Tris Base, Ethanol
P-W2-2 ----- Ethanol
P-EB1 ----- Tris-Base
P-EB2 ----- Tris-HCl, EDTA
P-EB3 ----- NaCl, Tris-Base, Isopropanol
Generic brand A (an "original equipment manufacturer" or "OEM" brand) and generic brand B (well-established brand) buffers and were purchased from the commercial vendors.
Culturing of Escherichia coli for miniprep: Luria–Bertani (LB, Biopolis Shared Facilities, BSF, A*STAR) with ampicillin (GoldBio, USA) used as growth medium. Previously made competent E. coli [4] were transformed with ampicillin resistant plasmids bearing antibody genes as previously described [5], and inoculated in LB ampicillin broth in overnight cultures at 37°C in a shaking incubator. The plasmids were used for miniprep, gel extractions, and PCR amplification. For comparisons, the same plasmids and bacterial cultures were used.
Establishing the OPT and HM buffers with generic brand A miniprep kit: To establish the best "home-made" (HM) proprietary buffers, we evaluated the solution by systematically displacing the buffers in kit A while following its protocol (Supplementary Material). For the selection of optimized (OPT) buffers, we chose the best buffers between our HM buffers and generic A. Comparisons were performed in triplicate minipreps, standardizing the use of 4 mL overnight E. coli culture, and 40 μl of buffer for elution. The HM and OPT buffers were selected based on DNA concentration, and A260/280 ratio (Supplementary Data)
Comparison of HM and OPT buffers on generic A and B miniprep kit: Plasmid extraction using HM buffer, OPT buffers, generic A and B plasmid extraction kit were each carried out in triplicates. All commercial kits were used according to the manufacturer's recommendations (see Supplementary Material). Comparison of HM and OPT buffers were performed according to the commercial protocol with the exception of varying the buffers on spin columns of both generic A and B.
Investigation of gel extraction buffers
Preparation of proprietary buffer for gel extraction: Proprietary gel dissolving buffers (P-QG2, P-QG3) for gel extraction were prepared with the following ingredients:
P-QG2 ----- NH2C(=NH)NH2 • HSCN, Tris-HCl, EDTA
P-QG3 ----- NH2C(=NH)NH2 • HSCN, C6H13NO4S • xH2O
Only the gel dissolution buffers were investigated as the other buffers were previously determined in the miniprep comparisons.
Electrophoresis and excision of gel fragment: 1% TAE agarose gel was used for running the same volume of plasmids in triplicates with 6x loading dye containing SYBR Green (Quintech Life Sciences Pte Ltd, Singapore). Gel bands were excised with a fixed volume gel cutter and weighed using Mettler Toledo analytical balance. The gel slices typically weighed between 200-300 mg.
Comparison of dissolving rate and DNA recovery of generic A and B gel extraction kits and proprietary buffers: Comparisons between gel extractions buffers from generic A, generic B, and the proprietary buffers (P-QG2, P-QG3) were performed in triplicates. Gel protocols for generic A and B (see Supplementary Material) were carried out according to respective manufacturer’s instructions with the exception of standardizing gel dissolution temperature to 60°C and elution of DNA at 35 μl. Gel extractions using proprietary buffers were carried out according to generic A protocol with the exception of varying the ratio of buffer to gel slice to 3:1 w/v ratio (according to generic B protocol). Time taken for the gel slices to dissolve completely were measured with a lab timer and analyzed statistically.
Optimization of PCR purification buffers
Polymerase chain reaction: PCR reactions of 325 μl were performed containing 6.5μl of Taq polymerase, 19.5 μl 30 mM MgCl2 and 32.5 μl of 10X PCR Buffer (Axil Scientific), 26 μl of 2 mM dNTPs mix (Quintech Life Sciences), 13 μl of reverse primer : OriPNrul R (5’-ATA TCT CGC GAA TGC TGG GGG ACA TGT ACC TC-3’), forward primer OriPNrul F (5’-CAC ACT CGC GAA GGA AAA GGA CAA GCA GCG AA-3’), template plasmid DNA, and 201.5 μl of HyClone water (Thermo Scientific, Cat no. SH30538.01). The amplicon oriP is ~1.9 kb. The completed PCR mix was transferred into PCR tubes of 25 μl aliquots and carried out in Arktik Thermal Cycler (Thermo Scientific) with the following profile: Initial denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 1 minute, annealing and extension at 71°C for 3 minutes; and final extension at 72°C for 10 minutes.
Comparisons of PCR purifications of generic A and B, and proprietary buffers: Generic A and B PCR purifications and selected proprietary buffers from miniprep (P-W1) and gel extraction (P-QG2) buffers were carried out in triplicates using the respective generic spin columns. A and B PCR purifications were performed according to the respective manufacturer’s recommendations (see Supplementary Material). The PCR purification using proprietary buffers were carried out using generic A’s protocol, with the exception of using 5:1 volume ratio of buffer to PCR reaction (according to generic brand B protocol).
DNA analysis
DNA concentration and A260/280 ratio were analysed spectrophotometrically using IMPLEN Nanophotometer P330 in triplicates. 1% TAE agarose gels were used to analyse quantity and quality of plasmid DNA extracted from the minipreps and PCR purifications. 10 μl of extracted/purified DNA from the above comparisons were loaded with 6x loading dye containing SYBR Green (Quintech Life Sciences) and analyzed using the RunVIEW electrophoresis apparatus (Cleaver Scientific).
Statistical analysis
Time taken for the gel dissolution, DNA concentration and A260/280 ratio from the nucleic acid extractions were analyzed using One-Way ANOVA and independent T-tests. Significance were deemed when p<0.05. All statistical analysis was performed using SPSS 17.0 (IBM).
Results and Discussion
From the systematic testing, we established a set of optimized (OPT) and completely "home-made" (HM) buffers for nucleic acid extraction and purification kits that are comparable to the two generic brands (A and B) in terms of plasmid yield and purity. Through the step-wise buffer substitution (Table 1), we found that P-P1, P-W2-1 and P-EB2 were buffers capable of obtaining high DNA yields.
On the equilibration of the spin columns (using generic brand A), the use of GA-BK buffer expectedly gave higher DNA yields than "no buffer" conditions (Table 1). Since the equilibration buffers contained Na+, a salt bridge could be formed, permitting DNA adsorption onto the silica particles [3]. We found that our P-BK buffer yielded less DNA compared to GA-BK (generic A) and P-BK2 buffer, as the latter two buffers had higher concentrations of Na+ (more than double the molarity), this demonstrates that high [Na+] allowed for effective equilibration.
Regarding the neutralization buffers, P-P3-2 buffer yielded significantly higher DNA than the P-P3-1 buffer (t (70) = 2.121, p=0.038, see Table 1). Since both P-P3-1 and P-P3-2 buffers acted to neutralize the alkaline lysis buffer, the resultant pH after neutralization would be lower for P-P3-2 due to its stronger acid component, supporting previous reports that lower pH (<7) facilitated better DNAsilica adsorption [3].
Amongst the wash buffers, P-W2-1 buffer had the highest DNA yield while P-W2-2 buffer had the lowest yields. From studying buffer recipes, we propose that the higher salt content in P-W2-1 increased the stringency of the column washes, removing nucleases more efficiently and preventing DNA degradation.
On the comparison of the elution buffers, P-EB2 was significantly better than P-EB1 (t (64) = 2.19, p = 0.032). As the only elution buffer with EDTA, Mg2+, a co-factor in many nucleases [6] would have been chelated. Since P-EB-2 also had a hundred-fold more Tris than P-EB1, there would be better pH buffering without producing free radicals that would otherwise speed up the auto-catalytic activity of DNA [6]. Comparatively, P-EB3 showed the lowest DNA yields (even below that of generic A buffer). Since isopropanol was a component, it is likely that DNA precipitation may have occurred, lowering yields.
As generic A and B buffer recipes were not known to us, we were unable to discuss the likely factors that contributed to the different DNA yields observed for the resuspension, lysis and pre-wash buffers (P1, P2, and W1, respectively).
With the HM and OPT buffer components determined, benchmarking was performed against the generic brand A and B miniprep kits. Generic brand B was specifically picked due to the company’s reputation in these kits. Our comparisons (Figure 1) found that the use of HM buffers on generic A columns did not perform better than generic A buffers. This was expected since some HM buffers (equilibration, lysis and neutralization) gave poorer yields than generic A counterparts (Table 1).
When OPT buffers were used on generic A spin columns (top panel of Figure 1, lane 2), and HM buffers on generic B spin columns (top panel of Figure 1, lane 6), we found that OPT buffers had higher yields than both generic A and B kits when using their respective buffers (top panel of Figure 1, lanes 1 and 4, respectively). HM buffers on generic B spin columns had the highest yields despite yielding the least DNA on generic A spin column. This shows that generic B spin columns had superior DNA binding capability, and that OPT buffers would give the best yields since they outperformed generic A buffers, which were in turn, superior to HM buffers (top panel of Figure 1). As the exact differences between generic A and B spin columns were unknown to us, we are unable to discuss this further.
On the fastest gel dissolution time, P-QG2 buffer showed the fastest average rate (211 secs), followed by P-QG3 (220 secs), generic B QG (234 secs), and generic A G-G1 buffer (354 secs; see Figure 2). ANOVA tests showed that the time differences were significant (F (3, 32) = 129.86, p=0.000).
On DNA recovery, P-QG2 had better or similar DNA recovery compared to both generic A and B buffers, respectively (Table 2), whereas P-QG3 buffer had lower yields than generic brand B kit despite being comparable to brand A. Investigations between our two proprietary buffers showed that P-QG2 had higher concentrations of guandidine thiocyanate (by almost 1 M). As a chaotropic agent that removes DNA binding proteins [7], the higher concentration of guanidine would have aided in better adsorption to the silica gels. Being similar to other chaotropic agents (e.g. potassium or sodium iodide), which are necessary for dissolving agarose gels [8], the higher concentrations of guanidine thiocyanate would also dissolve the agarose quicker. Thus, on the basis of timing and yields, P-QG-2 was chosen as the optimal buffer.
For PCR kits, we compared only the PCR binding buffers i.e. the optimized P-W1 and P-QG2 with generic A G1 and B’s buffers. ANOVA tests showed significant differences between the DNA recovered, F (5, 48)=261.72, p=0.000. It was observed that using generic B column, buffer P-W1 (P-W1-B in Figure 3A) obtained the highest DNA recovery, almost up to 30 ng/μL. On the contrary, the same P-W1 buffer in generic A column yielded only slightly above 20 ng/μL, thus supporting previous miniprep observations that generic B spin columns were superior with respect to DNA binding. Upon Normalizing the spin columns by comparing P-W1 on both A and B spin columns, buffers of both generic brands would generate similar yields.
Electrophoresis of the purified PCR products using the different spin columns revealed two distinct bands that corresponded to the OriP product and primer dimers. Interestingly, P-W1 removed primer dimers when used on generic A columns but not on generic B columns. This was likely due to the higher binding capability of generic B columns. Nonetheless, P-QG2 was clearly the better buffer as it yielded the highest intensity band regardless of the spin column used (Figure 3B and 3C).
Analysis of the P-QG2 and P-W1 recipes found that higher concentrations of guanidine (by ~1 M in P-QG2) resulted in better purification, which we propose to result from the release of polymerases from DNA, allowing their adsorption to the silica.
As a final comparison, we carried out trials comparing the full set of HM buffers and OPT buffers against both generic A and B (Figure 1). As can be observed, OPT buffers had the best yields regardless of the columns used, with HM buffers comparable to the commercial brands A and B.
The findings of the study allowed us to rely on more cost-effective columns without compromising experiments. Extending beyond the kits tested, the factors of these buffers also underline processes such as midi, maxi and giga scale DNA extractions, allowing labs to optimize their own cost-effective reagents by the addition of important chemicals to their existing buffers or kits (e.g. adding a Na+ column equilibration step to existing commercial kits). Through detailed analysis of buffer constituents, we were able to validate the importance of:
1) Na+ concentrations in column equilibration.
2) Importance of strong acids for low pH in the neutralization of cell lysis buffer.
3) High salt for higher stringency in column washes.
4) Tris and chelating agents to remove nuclease cofactors and pH buffers that would not generate free radicals.
5) High presence of chaotropic agents for faster gel dissolution and removal of interfering proteins for both gel and PCR kits.
We would like to thank Sir David Lane, Chandra Verma, Peck-Ting for their administrative support, without which the work would not be possible. We also thank Mr Keane MJ Lim for assisting in the formatting of the article. This work is mainly funded by Quintech Life Sciences Pte Ltd, with support from the Joint Council Office, Agency for Science, Technology, and Research, Singapore.
Competing Interests
This work was commissioned by Quintech Life Sciences Pte Ltd to explore the factors to making better buffers, which may be made commercially available. There are no other competing interests.

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