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New approaches in the binding of DNA for clinical applications.

DNA, the template coding for the proteins associated with all cellular functions (1), interests biochemists and, more recently, molecular biologists. With emerging opportunities in gene therapy (2), an understanding of the host genome is necessary if the defective gene is to be replaced by a corrected one. This requires not only the therapeutic gene but also a probe for identifying patients expressing the defective gene and subsequently monitoring the efficacy of the treatment. This aspect of gene screening is a growing area in molecular diagnostics and may depend on a need to isolate host genomic DNA from clinical samples. Similarly, in genetic screening in forensics and human reference data, the move to construct databases (3) produces a requirement to isolate the host genomic DNA from a biological sample such as blood, bacteria, and cells in tissue culture. Isolation of DNA involves four discreet stages: (a) cell disruption to release nuclear material, (b) selective purification of the DNA-containing fraction, typically using precipitation of contaminants and digestion of RNA, (c) adsorption of the DNA to a solid support, and (d) selective desorption of the DNA and elution from the matrix. The eluted DNA may then be used for applications such as PCR, sequencing, or other analytical techniques.

One of two generic adsorption processes typically is used for DNA isolation following the partial purification mentioned above: (a) hydrogen-bonding interaction under chaotropic conditions to an underivatized hydrophilic matrix, typically silica (4, 5), or (b) ionic interaction under aqueous conditions to an anion exchanger.

We have recently reported the development and application of Magarose[R], an agarose bead containing a paramagnetic component, which can be used in its anion-exchange form, DEAE-Magarose, for isolation of plasmid and genomic DNA from a nucleic acid preparation (6). In the present study we describe the isolation of plasmid DNA to new high surface area nonporous particulate silica matrix and also to a new laminar format silica disc.

Silica matrix and silica discs were obtained from Whatman. A silica spin kit for plasmid DNA minipreparation and a silica spin disc kit for plasmid DNA minipreparation containing silica matrix or discs respectively were from Biometra.

Escherichia coli JM109 cells expressing the plasmid pBluescript were grown to late log phase in Luria-Bertani broth containing 100 mg/L ampicillin. The bacterial cells were harvested from 1.5 mL of cell culture by centrifugation at 75008 for 1 min. The pellets were resuspended in 0.05 mol/L Tris-HCl buffer (pH 7.5) containing 0.01 mol/L EDTA and 50 mg/L ribonuclease A (200 [micro]L). Cell lysis was performed by gently mixing the resuspended cell pellet with 0.2 mol/L NaOH containing 10 g/L sodium dodecyl sulfate (200 [micro]L). Genomic DNA and other contaminants were precipitated by addition of 0.75 mol/L potassium acetate (pH 4.6) containing 4 mol/L guanidine hydrochloride. The mixture was centrifuged at 75008 for 5 min to sediment the precipitated protein, cell debris, and denatured chromosomal DNA.

A silica spin disc filter unit or a spin filter containing silica matrix suspended in 0.75 mol/L potassium acetate (pH 4.6) containing 4 mol/L guanidine hydrochloride (50 [micro]L) was placed in a 1.5-mL microcentrifuge tube, and the plasmid DNA-containing solution was applied to the spin filter units. The tubes were centrifuged for 30-60 s, and the filtrate was discarded. The silica matrix or disc was washed with 0.75 mol/L potassium acetate (pH 4.6) containing 4 mol/L guanidine hydrochloride (500 [micro]L). After centrifugation, the filtrates were discarded. Residual chaotrope was removed by washing the silica matrix or disc with 250 [micro]L of ethanol in 250 [micro]L of 0.1 mol/L Tris-HCl buffer, pH 7.5, containing 0.02 mol/L EDTA and 0.4 mol/L NaCl. After centrifugation for 60-120 s, the filtrates were discarded. Plasmid DNA was eluted from the silica matrix or disc with either 0.01 mol/L Tris-HCl buffer, pH 8.0, containing 0.001 mol/L EDTA (100 [micro]L) or [H.sub.2]O (100 [micro]L) and was collected by centrifugation.

The isolation of pBluescript plasmid DNA from E. coli JM109 cells is summarized in Table 1 and compared with data obtained by use of the anion exchanger, DEAE-Magarose (6). The data demonstrate that plasmid DNA can be successfully isolated from a bacterial cell culture after alkaline lysis, where genomic DNA and proteins are denatured and RNA is hydrolyzed. The released plasmid DNA is recovered from the lysate under chaotropic conditions using both forms of our silica matrix. The eluted pBluescript gave a read length of at least 900 bases after sequencing using an ABI Prism Model 377 DNA Sequencer, confirming the high quality of the purified DNA. This is reflected by the high [A.sub.260]/[A.sub.280] ratio (Table 1) and was confirmed by agarose gel electrophoresis.

The silica matrix offers a degree of flexibility in Optimizing the mass of matrix required to bind sufficient DNA for the application, whereas the silica discs provide a laminar format perhaps more suitable for molecular diagnostic device development because of their ease of handling. Furthermore, the silica matrix used in this study is nonporous, which not only reduces the likelihood of contaminant carryover from step to step, a consideration when using conventional porous silica materials, but also permits small elution volumes with high recovery.

DNA isolation is gaining importance in the fields of molecular diagnostics. In the present study, we report two approaches to isolation of high-quality DNA from a prokaryotic source in a relatively short time. By way of comparison, we report a similar purification by ion exchange using DEAE-Magarose (6). Although this yields DNA of greater purity, it is a slower process, and the DNA eluted requires desalting by precipitation before use. Depending on the intended use of the DNA, these issues may be of importance in selecting the most appropriate technique. Although the applications presented here are not clinical examples, they serve the purpose of demonstrating proof of concept for this type of diagnostic application. We feel that the adsorptive methods described here could be considered when developing a molecular diagnostic test protocol, and depending on the process requirements, an interaction with silica under chaotropic conditions using either particulate or laminar matrices may be suitable.

References

(1.) Stryer L. Biochemistry, 2nd ed. San Francisco: Freeman, 1981:511pp.

(2.) Wils P, Escriou V, Warnery A, Lacroix F, Lagneaux D, Ollivier M, et al. Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography. Gene Therapy 1997;4:323-30.

(3.) Chadwick N, Bruce I, Davies M, van Gemen B, Schukkink R, Khan K, et al. A sensitive and robust method for measles RNA detection. J Virol Methods 1998; 70:59-70.

(4.) Vogelstein B, Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A 1979;76:615-9.

(5.) Melzak KA, Shenvood CS, Turner RFB, Haynes CA. Driving forces for DNA adsorption to silica in perchlorate solutions. J Colloid Interface Sci 1996; 181:635-44.

(6.) Levison PR, Badger SE, Dennis J, Hathi P, Davies MJ, Bruce IJ, Schimkat D. Recent developments of magnetic beads for use in nucleic acid purification. J Chromatogr A 1998;816:107-11.

Peter R. Levison, [1] Jon W. Dennis, [1] Kevin D. Jones, [1] Richard W. Philpott, [1] Susan L. Taylor, [2] and Volker Grimm [3]

([1] Whatman International Ltd., Springfield Mill, James Whatman Way, Maidstone, Kent, ME14 2LE, United Kingdom; [2] Whatman Inc., Clifton, NJ 07014; [3] Biometra GmbH, D-3400 Gottingen, Germany; * author for correspondence: fax 44-1622-674490, e-mail peterl@ whatman.co.uk)
Table 1. Isolation of pBluescript plasmid DNA from E. coli
JM109 cell culture.

 Culture
 volume, mL Yield [+ or -] SD,
Matrix [micro] g (n)

Silica matrix 1.5 10.73 [+ or -] 0.50 (3)
Silica disc 1.5 14.57 [+ or -] 0.91 (3)
DEAE-Magarose 1.5 8.22 [+ or -] 0.73 (5)

 [A.sub.260]/[A.sub.280]
 ratio Extraction
Matrix time, min

Silica matrix 1.80 40
Silica disc 1.80 40
DEAE-Magarose 1.94 90
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Title Annotation:Poster Sessions
Author:Levison, Peter R.; Dennis, Jon W.; Jones, Kevin D.; Philpott, Richard W.; Taylor, Susan L.; Grimm, V
Publication:Clinical Chemistry
Date:Sep 1, 1998
Words:1349
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