Proprep™ for Large Scale Extraction and Purification of Clinical-Grade Plasmid DNA for DNA Vaccines.

Proprep™ for large scale extraction and purification of clinical-grade plasmid DNA for DNA vaccines.

Advances in DNA vaccination and gene therapy have created a need for the large scale extraction and purification of clinical-grade plasmid DNA. The growing demand for large-scale plasmid production for DNA vaccines poses new demands on the purification of plasmid DNA. The ProPrep™ strategy does not rely on bind/elute protocols, consequently achieving well to well consistency. Large-scale production of DNA requires an efficient production method of plasmid DNA (pDNA) for the extraction and purification of clinical-grade plasmid DNA.

In a published article by Huang, G.M., et al published in Analytical Biochemistry, titled, "A High-Throughput Plasmid DNA Preparation Method" researchers used ProPrep™ BAC 96 for plasmid DNA preparation based on alkaline denaturation in a 96-well format. Purification is done by ProCipitate™ in 96-well micro filters. Using this method scientists optimized the right bacterial strain for plasmid yield and did further analysis of different isoforms. The yield and quality of DNA are sufficient for restriction digestion, radioactive sequencing, and automated fluorescent sequencing to ultimately develop new medicines for gene therapy. The steps involve an alkaline lysis miniprep to screen bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. Next producing milligrams of plasmid DNA and purifying the crude lysate by centrifugation. 96 samples can be purified from crude tissue digests and are produced in microtitre plate format to allow efficient downstream processing of samples.

What is special about Proprep™ method?

• The ProPrep™ strategy does not rely on bind/elute protocols, consequently achieving well to well consistency. Smaller DNA molecules are difficult to elute because such molecules bind more tightly. Larger molecules may bind to more than one particle and get sheared during manipulation of the matrix with DNA bound to it. Proprep™ avoids such bind/elute protocols.
• Proprep™ has a scaled down starting culture, 250 ul (2xYT broths), which makes ProPrep™ Plasmid 60x96 ideal for low cost mini-prep template DNA for high copy plasmids.
• Using the simple method for plasmid purification, ProPrep™ does not require HPLC or ultracentrifuges.
• Isolation of plasmid DNA with phenol/cholorform is environmentally unfriendly. ProCipitate™ is non-hazardous and can replace phenol/chloroform with the additional benefits of solid-phase suspensions. Phenol and ethidium bromide are toxic and so the ProPrep™ ProCipitate™ method does not use them.
• For isolation of plasmid for DNA-based vaccines, the Proprep™ method removes impurity of residual host-cell RNA in the production of clinical-grade plasmid DNA.

What are advantages of using Proprep™?

• Advances in research experiments such as the uptake and prolonged in vivo expression of transgenes inserted on a plasmid injected into the leg muscle of mice created interest in gene therapy and the design of novel DNA-based vaccines. An example of plasmid DNA’s clinical application is use of a plasmid encoding for VEGF, a vascular endothelial growth factor that has been shown to promote revascularization in ischemic limbs and hearts of patients.
• Proprep™ provides a methodology to prepare scalable, clinical grade DNA plasmid lots which are substantially free of host cell protein, host cell endotoxin, genomic DNA, genomic RNA and plasmid degradates such as linear and open circle forms. The purified DNA is ready for the downstream applications such as sequencing, sub-cloning, gene expression, and transfection.
• Using Proprep™, isolate nucleic acid including high molecular weight DNA and use it in a number of molecular biology techniques, including polymerase chain reaction , DNA hybridization, restriction enzyme digestion, DNA sequencing, and array-based experiments.
• Proprep is robust, high throughput compatible, has a low degree of technical difficulty and is low cost making it the ideal plasmid DNA preparation method of choice in both expressed sequence tag (EST) and genome sequencing projects.

What are tips for using Proprep™?

• Plasmid DNA isolation (i.e., plasmid preps, mini-preps, rapid DNA preps, among other procedures) is efficient using Proprep™ from a DNA source, e.g., blood, saliva, bacterial cultures, etc.
• Do not vortex or mix the sample vigorously because the genomic DNA is easy to break, and broken genomic DNA can be small enough to re-anneal and go into solution with the plasmid.
• The simple ethanol procedure should be suitable for most typical small plasmids, 2-3 kb and for techniques such as restriction mapping. For larger plasmids, consider the isopropanol procedure for highest yields and purity. Alcohol precipitation adapted to 96 well filtration processes, in lieu of centrifugation are available.

References:

• Huang, G.M., et al, A High-Throughput Plasmid DNA Preparation Method, Analytical Biochemistry, 223:35-48, 1994.
• Kelley, J.M., et al, High-Throughput Direct End Sequencing of BAC Clones, Nucleic Acids Reseach, Vol. 27, No. 6: 1539-1546, 1999.
• Reddy, O.U.K., et al, New Dinucleotide and Trinucleotide Microsatellite Marker Resources for Cotton Genome Research, Journal of Cotton Science, 5:103-113 (2001).
• Klein, R.K., et al, High Throughput BAC DNA Isolation for Physical Map Construction of Sorghum, Plant Molecular Biology Reporter, Kluwer Academic Publishers, 1998.
• Bruce, D.C., et al, BAC Library End Sequencing in Support of Whole Genome Assemblies,
• Poster DOE Joint Genome Institute and Center for Human Genome Studies, Los Alamos
• National Laboratory.
• Bolivar, et al., Construction and Characterization of New Cloning Vehicles, Gene 2:95-113 (1977)
• Bywater, et al., A Novel Chromatographic Procedure for Purification of Bacterial Plasmids, Analytical Biochemistry 132:219-224 (1983)
• Godson, et al., A Simple Method of Preparing Large Amounts of .PHI.X174 RF I Supercoiled DNA, Biochimica et Biophysica Acta 299:516-520 (1973)
• Gorman, et al., High Efficiency DNA-Mediated Transformation of Primate Cells, Science 221:551-553 (1983)
• Gorman, et al., The Rous Sarcoma Virus Long Terminal Repeat is a Strong Promoter when Introduced into a Variety of Eukaryotic Cells by DNA-mediated Transfection, Proc. Nat. Acad. Sci., USA 79:6777-6781 (1982)
• Harding, et al., Rapid Isolation of DNA from Complex Biological Samples Using a Novel Capture Reagent- Methidium-Spermine-Sepharose, Nucleic Acids Research 17:6947-6958 (1989)
• Hillen, et al., Preparation of Milligram Amounts of 21 Deoxyribonucleic Acid Restriction Fragments, Biochemistry 20:3748-3756 (1981)
• Hines, et al., Large-Scale Purification of Plasmid DNA by Anion-Exchange High-Performance Liquid Chromatography, BioTechniques 12: 430-434 (1992)
• Johnson, et al., Large-Scale Isolation of Plasmid DNA and Purification of λ Phage DNA Using Hydroylapatite Chromatography, Analytical Biochemistry 132:20-25 (1983)
• Kozak, M., An Analysis of 5'-Noncoding Sequences from 699 Vertebrate Messenger RNAs., Nucleic Acids Res. 15:8125-8148 (1987)
• Mahon, et al., Prenatal Lethality in a Transgenic Mouse Line is the Result of a Chromosomal Translocation, Proc. Nat. Acad. Sci., USA 85:1165-1168 (1988)
• Marko, et al., A Procedure for the Large-Scale Isolation of Highly Purified Plasmid DNA Using Alkaline Extraction and Binding to Glass Powder, Analytical Biochemistry 121:382-387 (1982)
• McClung, et al., Purification of Plasmid DNA by Fast Protein Liquid Chromatography on Superose 6 Preparative Grade, Analytical Biochemistry 177:378-382 (1989)
• Nabel, et al., Direct Gene Transfer with DNA-Liposome Complexes in Melanoma: Expression, Biologic Activity, and Lack of Toxicity in Humans, Proc. Nat. Acad. Sci., USA 90:11307-11311 (1993)
• Overbeek, et al., Tissue-Specific Expression in Transgenic Mice of a Fused Gene Containing RSV Terminal Sequences, Science 231:1574-1577 (1986)
• Raymond, et al, Large-Scale Isolation of Covalently Closed Circular DNA Using Gel Filtration Chromatography, Analytical Biochemistry 173:125-133 (1988)
• Sambrook, et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). pp. 1.21-1.52
• Summerton, et al., A Rapid Method for Preparation of Bacterial Plasmids, Analytical Biochemistry 133:79-84 (1983)
• Swanstrom, et al., Nucleotide Sequence of Cloned Unintegrated Avian Sarcoma Virus DNA: Viral DNA Contains Direct and Inverted Repeats Similar to Those in Transposable Elements, Proc. Nat. Acad. Sci., USA 78:124-128 (1981)
• Thompson, A Review of High-Performance Liquid Chromatography in Nucleic Acids Research. III. Isolation, Purification, and Analysis of supercoiled Plasmid DNA, BioChromatography 1:68-80 (1986)
• Thompson, et al., Purification of Nucleic Acids by RPC-5 Analog Chromatography: Peristaltic and Gravity-Flor Applications, Methods in Enzymology 100:368-399 (1983)
• Westphal, et al., Promoter Sequences of Murine 댒(I) Collagen or of Avian Sarcoma Virus Genes Linked to the Bacterial Chloramphenicol Acetyl Transferase Gene Direct Tissue-specific Patterns of Chloramphenicol Acetyl Transferase Expression in Transgenic Mice, Cold Spring Harbor Symp. Quant. Biol. 50:411-416 (1985)
• Willis, et al., Prep-A-Gene: A Superior Matrix for the Purification of DNA and DNA Fragments, BioTechniques 9:92-99 (1990)
• Yamada, et al., A New Method for Extracting DNA or RNA for Polymerase Chain Reaction, Journal of Virological Methods 27:203-210 (1990)