The use of PCR for increasingly large-scale clinical and research applications requires an effective method of sample processing. Unfortunately, isolation of PCR-quality DNA is time-consuming and not easily adaptable for automation. An alternative to DNA purification would be to lyse the cells using alkali and then take an aliquot of the released DNA for PCR amplification. However, published methods employing alkaline lysis require additional steps, including neutralization or other manipulation of the lysates prior to PCR (1,2,3,4).
We sought to develop an alkaline lysis reagent that would allow a wide variety of biological samples to be used directly in PCR, without further neutralization or DNA isolation steps. We found that polyethylene glycol (PEG) at a high concentration (>50%) and at alkaline pH meets these criteria. This solution, which we will refer to as the alkaline PEG reagent, consists of 60% PEG 200 (tetraethylene glycol; Sigma-Aldrich, St. Louis, MO, USA) and 20 mM KOH and has a pH of 13.3-13.5. The procedure for processing samples with the alkaline PEG reagent is extremely simple; a step-by-step protocol is listed in (Table 1).Table 1. Composition and Protocol for Alkaline PEG Reagent
PEG, polyethylene glycol.
As the examples in (Figure 1) show, PCR can be performed on microorganisms and solid or liquid samples of human, animal, or plant origin that have been prepared with the alkaline PEG reagent. Data are shown for PCRs performed with human saliva, human blood, rat liver, wheat sprouts, and Citrobacter braakii. Notably, as shown in lane 2 of (Figure 1), successful PCR amplification can be obtained from human blood stored in the reagent for 3 months at room temperature. Lane 4 demonstrates successful amplification of a 5-kb product; more detailed studies have determined that fragments up to 8 kb can be amplified (data not shown). Lysates in alkaline PEG reagent also support DNA amplification in multiplex PCR (lane 3). Sequencing of the amplified 194-bp human COX-2 and 707-bp human cfos DNA fragments (lane 3) confirmed that the alkaline PEG lysate supports amplification of accurate copies of DNA fragments (data not shown).Figure 1.
A pH greater than 13 is required to keep DNA in solution in the alkaline PEG reagent and for the optimal sample processing. At a lower pH, PEG may precipitate DNA (5). The ability to achieve a pH greater than 13 using 20 mM KOH results from properties of glycols in aqueous solution. We found that at alkaline pH, concentrated glycols significantly contribute to the alkalinity of a solution ((Table 2)). The 20 mM KOH, 60% PEG 200 solution has a pH of 13.45, while 20 mM KOH alone has a pH of 12.48. Thus, 60% PEG 200 produces an approximately 10-fold increase in the concentration of OH–. As a result, the alkaline PEG reagent has an alkalinity equivalent to that of 200 mM KOH (pH 13.5). The alkaline effect of PEG is concentration-dependent. The 10-fold dilution of the alkaline PEG reagent with water decreases its pH by about 2 U (20-fold decrease in OH– concentration). This drop in pH is sufficient to bring the diluted reagent within buffering capacity of a standard PCR buffer. For instance, when the alkaline PEG reagent is diluted 10-fold in a buffer containing 10 mM Tris, pH 8.3 (e.g., Sigma-Aldrich PCR buffer), its pH decreases to 8.63, well within the effective range for PCR. The presence of a biological sample, such as blood, further neutralizes the alkaline PEG reagent down to pH 8.48. In contrast, a PCR mixture containing the blood lysate in 200 mM KOH has a pH of 11.86 and must be adjusted before use in PCR.Table 2. Concentration-Dependent pH Changes of PEG Solution
Aliquots of alkaline PEG reagent (20 mM KOH-60% PEG) were diluted 10-fold with water or PCR mixture (10 mM Tris-HCl, pH 8.3, at 25°C, 50 mM KCl, 1.5 mM MgCl2 and 0.001% gelatin; Sigma-Aldrich PCR buffer). Ten microliters of human whole blood were lysed with 190 µL of alkaline PEG reagent or 200 mM KOH. The blood lysates were incubated for 15 min before dilution with the PCR mixture. The pH of the solutions was recorded before and after the dilution. PEG, polyethylene glycol.
The alkaline effect is observed to varying degrees with other glycols including polypropylene glycol, but is not observed with ethylene glycol monomer and polyalcohols such as glycerin. We empirically found that PEG 200 is optimal for the alkaline PEG reagent. It is an organic solvent (melting point at -65°C) with a relatively low viscosity (50 centi Poise at 25°C). As an organic solvent, PEG 200 stabilizes the reagent by reducing the solubility of CO2, which would otherwise have a neutralizing effect on the reagent over time. In our tests, the alkaline PEG reagent stored at room temperature in a container that is opened at least weekly remained functional for PCR applications for 1.5 years.
A molecular mechanism of the alkaline effect of PEG remains to be elucidated. The alkaline effect of PEG can be explained by its interaction with water, at alkaline pH, which results in binding H+, dissociation of water, and increase in the concentration of free OH–. Triethylene glycol and higher PEG polymers in aqueous solutions are characterized as nonionic kosmo-tropes, water-structure makers (6,7). We propose that at alkaline pH, PEG 200, and higher PEG polymers acquire chaotropic properties of water-structure breakers.
Processing samples in alkaline PEG reagent may inactivate common pathogens. We found that Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus did not survive a 15-min exposure to the alkaline PEG reagent at room temperature. The inactivation of Staphylococus aureus required 60 min and Enterococcus faecium over 120 min at room temperature. At 90°C, S. aureus and E. faecium did not survive a 10-min exposure to the reagent. Therefore, the inactivation of infectious material by the alkaline PEG reagent must be determined for each specific pathogen under the conditions employed and heating may be required to inactivate some pathogens. We did not test the survival of viruses in the reagent. However, the high pH of the alkaline PEG reagent should favor viral inactivation by hydrolysis of RNA viruses and denaturation of DNA viruses (8).
In conclusion, the alkaline PEG reagent is a universal reagent that can be used to process a wide range of biological samples for PCR assays in forensic, clinical diagnostic, biotech-nological, and biomedical research applications. The alkaline PEG reagent can also be used to process formalin-fixed, paraffin-embedded tissue sections (manuscript in preparation). Sample processing in alkaline PEG is fast, and the resulting lysates can be used directly for PCR without additional manipulation. A 35-cycle PCR with the alkaline PEG lysate can detect as little as 10 pg DNA (data not shown). The alkaline PEG reagent has been tested and found compatible with the following PCR kits: AccuPrime™ kits (Invitrogen, Carlsbad, CA, USA), Sigma Taq kit (Sigma-Aldrich), and Ex Taq™ kit (Takara Bio, Seta, Japan). The use of the alkaline PEG reagent simplifies PCR diagnostics, enables the effective processing of a large number of samples at minimal costs, and makes possible the automation of sample processing for PCR-based robotic systems.
We thank Drs. Judith Heiny and William Wilfinger for helpful discussions and Q Laboratories (Cincinnati, OH, USA) for performing tests with pathogens.
P.C. and M.R. are employed by Molecular Research Center, which has applied for U.S. and international patents that would establish intellectual property rights and restrict commercial use of the described reagent.