to BioTechniques free email alert service to receive content updates.
Regeneration of commercial nucleic acid extraction columns without the risk of carryover contamination
 
Nagadenahalli B. Siddappa, Appukuttan Avinash, Mohanram Venkatramanan, and Udaykumar Ranga
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
BioTechniques, Vol. 42, No. 2, February 2007, pp. 186–192
Full Text (PDF)
Abstract

Nucleic acid extraction is a basic requirement in a molecular biology laboratory. In terms of purity and yield, commercial nucleic acid extraction columns are superior; however, they are expensive. We report here an efficient strategy to regenerate diverse commercial columns for several rounds without altering the binding capacity of the columns or changing the properties of the nucleic acids purified. Plasmids purified with regenerated columns were functionally identical in super-coiled nature, restriction analysis, expression of the encoded reporter genes, or amplification of the viral RNA in real-time PCR. To ensure that the regenerated columns were free of the residual DNA, we used two different plasmids with different drug-resistance markers. By colony plating and PCR amplification of the encoded genes, we show that the regeneration process is absolute. Using radiolabeled DNA, we demonstrate that DNA exposed to the regeneration reagent is fragmented to molecular weight below 36 bp. Our data collectively prove regeneration of the commercial columns without the concern of carryover contamination. A procedure to permit safe and efficient regeneration of the commercial columns is not only of great advantage to extend the lifetime of these columns but also makes them commercially more affordable, especially in a resource-poor setting.

Introduction

Nucleic acid extraction is one of the most basic requirements in a molecular biology laboratory. The diverse nucleic acid extraction protocols by and large fall into one of three categories: (i) the classic extraction methods; (ii) unmodified silica resin-dependent protocols; and (iii) commercial column-based strategies. The classic alkaline lysis method (1) and the phenol chloroform extraction strategy (2) are highly popular, as they are the least expensive and do not require sophisticated equipment. These protocols, however, are associated with serious practical limitations, including the quality of the DNA extracted and the time required for processing a large number of samples. Furthermore, these procedures are not preferred for amplification-based clinical diagnosis, considering the impending risk of contamination (3,4). A wide variety of protocols have been developed using native unmodified silica resin (5,6,7,8,9,10,11) or its derivatives, such as diatomaceous earth (12) or pumice (13) for preferential isolation of nucleic acids. Although the quality and quantity of the nucleic acids extracted using the crude silica are superior to the conventional techniques, they are certainly not suitable for a range of applications, such as in vitro transcription/translation, microinjection, and immunization (8). When purity of the nucleic acids is an important consideration, commercial nucleic acid extraction columns have no substitute. A variety of commercial nucleic acid extraction columns available from several vendors by and large use a similar strategy to purify nucleic acids by exploiting the negative charge of the phosphate backbone. Many of the commercial nucleic acid extraction columns are essentially silica-based; however, the silica resin or membranes are chemically modified by attaching strong anionic groups such as the diethylaminoethyl (DEAE) moiety. The high surface density of the anion groups on such chemically modified silica resins makes the resins efficient and highly selective for nucleic acids in the presence of high salt concentration and pH conditions. Commercial nucleic acid extraction columns, therefore, ensure the highest level of nucleic acid recovery and purity with little technical complication. Commercial nucleic acid extraction columns, however, are expensive and are disposed of after a single use. Considering the cost factor, we set out to develop a procedure to regenerate the commercial nucleic acid extraction columns. We found that incubation of used commercial nucleic acid extraction columns in 1 M HCl for 24 h can efficiently eliminate bound DNA and regenerate them for a fresh round of use. Applying this strategy, the commercial nucleic acid extraction columns can be regenerated many times without a loss in the binding capacity and, importantly, without the possibility of carryover contamination.

Materials and Methods

Regeneration of the Commercial Columns

Commercial nucleic acid extraction columns after one round of use were stored in 1 M HCl for a specified period or indefinitely. Before use, the columns were removed from the acid, rinsed thoroughly in sterile distilled water, and equilibrated with Buffer QBT (Qiagen, Valencia, CA, USA) or an equivalent. The volume of water and buffer used to wash the columns typically depended on the nature and the capacity of the columns. For Qiagen-tip 20 columns (Qiagen), for instance, we typically used 5 mL sterile distilled water per wash for five rounds and 5 mL Buffer QC (Qiagen) for one round to regenerate the columns.

Transfection of the Mammalian Cells and Reporter Gene Analysis

HEK 293 cells were transfected with plasmid expression vectors using a standard calcium phosphate method (14). Briefly, cells in 12-well plates were transfected with a total of 1.1 µg plasmid DNA, consisting of 1 µg reporter vector and 0.1 µg pCMV-β-galactosidase expression vector—the latter included in all the transfections to serve as an internal control for the transfection efficiency. CEM-GFP cells were electroporated using the Gene Pulser® II system (Bio-Rad Laboratories, Hercules, CA, USA) at 240 V and 950 µF capacitance with a total of 5 µg DNA consisting of a mixture of 4 µg Tat-expression vector, pCMV Tat, and 1 µg cytomegalovirus (CMV)-β-galactosidase plasmid. Gene expression from the transfected cells was monitored starting from 24 h after transfection up to 72 h. Quantitation of the reporter genes, including the secreted alkaline phosphatase (SEAP), green fluorescent protein (GFP), β-galactosidase, and the viral antigen p24 was performed as described previously (15).

  1    2    3    4