ProCipitate™ Superior Substitute to Phenol/Chloroform for DNA Isolation & Protein Extraction

Introduction

There are many DNA, RNA, and nucleic acid preparation products that serve the market well for most applications. Most of these products in some way utilize a bind/wash/elute strategy adopting surface reagents such as silica & metallic oxides and similar solid-phase chemistries. However, there still remain situations whereby these products are not optimal and for these applications Biotech Support Group supplies another option, based on itsProCipitate™ and related kit products reviewed here. The ProCipitate™ strategy is opposite to common prep strategies, as instead of binding nucleic acids, the protein is efficiently depleted with no interaction with the soluble nucleic acids. In this way it is characteristic of phenol/chloroform, a gold standard for nucleic acid preparation, but without the handling hazards and volatility. ProCipitate™ is offered in a variety of kits, providing all necessary buffers and accessories for different applications. A new kit, ProCipitate™ SF Kit, features a convenient spin-filter format for post-separations clean-up and categorization of journal reference applications for easy protocol development.

ProCipitate™ is a unique protein depletion reagent developed from patented solid-phase polyelectrolytes. These elastomeric polymer suspension reagents are prepared in an extended state due to strong electrostatic repulsion of the repeating polymeric acid groups. Upon interaction with proteins, salt bridges form and the resultant complex collapses to a lower energy state, expelling water much like dehydration processes taking place within solvent precipitations. Consequently, aggregation of proteins is strongly promoted and occurs even in high ionic strength or surfactant containing solutions. Most importantly, nucleic acids remain unreacted and are quantitatively recovered in solution.

In this way, ProCipitate™ is characteristic of phenol/chloroform separation, a long established benchmark for nucleic acid isolation. However, ProCipitate™ is non-volatile, non-hazardous, and has the additional benefits of solid-phase suspensions; that is the adaptability to filtration and automation.

ProCipitate™ and related kit products have been on the market for close to 20 years being used throughout the Human Genome Sequencing Project. It is routinely used and cited in protocols for improving the yield consistency and protein depleted quality of DNA. Such improvements have been cited in sequence and PCR quality for a variety of applications, most notably in the template preparation of large insert plasmids (cosmids & BACs) and PCR suitability for infectious agents from large volumes and from paraffin-embedded tissues.

Sample Size	ProCipitate™ Typical Usage
1 ml Yeast Culture Genomic DNA	200 ml
Mouse Tail Genomic DNA	200 ml
4 mm Plant Leaf	200 ml
1.5 – 2.0 ml culture BAC Preps	80 ml
5mm paraffin-embedded tissue	200 ml
250 ml culture Plasmid Preps	20 ml
200 ml Large Scale BAC Preps	5 ml
Dried Blood Card or ~ 40 ml Whole Blood	200 ml
200ml lysed cell pellet	200 ml

ProCipitate™ can even be used for enrichment of other macromolecules including proteoglycans, polysaccharides, glycolipids, and highly substituted polymer conjugates (i.e., PEG), which serve to mask salt bridge formation and retain solubility. A full list of references is provided at the end of this review.

Product	Size	Item No.
ProCipitate™	30 ml	P0050-30
ProCipitate™	100 ml	P0050-100
ProCipitate™	500 ml	P0050-500

ProCipitate™ SF Kit
200ml DNA Prep Microfuge Spin-Filter kit with ProCipitate™

Adapt ProCipitate™ to any low volume application for

• Pathogen and infectious disease testing.
• Tissue and paraffin-embedded tissues

Sample Size	ProCipitate™ Volume
1 ml Yeast Culture Genomic DNA	200 ml
Mouse Tail Genomic DNA	200 ml
4 mm Plant Leaf	200 ml
5mm section paraffin-embedded tissue	200 ml
Dried Blood Card or ~ 40 ml Whole Blood	200 ml
200ml lysed cell pellet	200 ml

500 ml microfuge spin-filters add convenience to all ProCipitate™ protocols. These are now included in the ProCipitate™ SF Kit. Protocols suggested process 200 ml lysed cell pellets, paraffin-embedded tissue or 40 ml whole blood.

Product	Size	Item No.
ProCipitate™ SF Kit	25 preps	PSF-25
ProCipitate™ SF Kit	100 preps	PSF-100

ProCipitate™ is packaged within application specific kits – called ProPrep™, which include the lysis and resolubilization buffers. These include:
ProPrep™ Genomic 96/100 & ProPrep™ BAC Mini

ProPrep™ Genomic 96/100
Whole Blood DNA Purification System with ProCipitate™

• PCR can be achieved from as little as 1 ng of template DNA
• Adaptable to 96-well direct lysis of 50µl whole blood
• Does not require reduction to buffy coat, filtration compatible, no precipitation
• Customize a massive PCR, SNP or NGS strategy

ProPrep™ Genomic 96/100 is a complete nucleic acid purification system based upon the unique protein extraction reagent,ProCipitate™. The basic protocol includes one step chaotrope lysis of whole blood (no buffy coat reduction), followed by removal of contaminating proteins and heme with ProCipitate™.

The ProPrep™ Genomic 96/100 permits the user to customize a massive PCR, SNP or NGS strategy without regard to collecting impractical quantities of whole blood. The isolated DNA is of the highest quality, and PCR can be achieved from as little as 1 ng of template DNA. This means that over 1,000 PCR reactions can be obtained from one, 50 µl whole blood sample.

Options – After separation, the purified DNA is contained within the lysis buffer. The DNA can then be either alcohol precipitated or simply diluted using the “Dilution Protocol” described below, to eliminate inhibitory effects of the lysis buffer. Note-protocols for separating the polymer-protein composite can either be done by centrifugation or filtration. 96-well plates are not stocked but recommendations for suitable suppliers can be made. The following protocol describes a 96-well filtration protocol.

• In a 96 deep well plate, to 50 ml of whole blood, add 100 ml of GL1D buffer to lyse the cells. Tape seal the plate and vortex for 30 seconds. Incubate 10 minutes at 65ºC and vortex again briefly.
• Shake ProCipitateä well to resuspend solid-phase. Using wide bore pipette tips, add 250 ml ofProCipitateä to each well, mix by pipetting up and down 8-10 times to insure that each sample is homogeneous. Allow to stand for 5 minutes.
• Transfer each sample to the corresponding well of the 96-well filter, and vacuum to collect filtrate, about 10-15 minutes. The collected supernatant contains the DNA.

Dilution and PCR – No alcohol precipitation

The volume recovered after filtration was approximately 250 ml. A 1:50 dilution was made and PCR was performed from 10 ml aliquots. Thus the number of projected PCR reactions from the final volume is 1,250.

50 ml of whole blood contained
» 1,270 ng DNA; final diluted volume
= 12,500 ml, contained 1,270 ng DNA
or 0.1 ng/ml; 10 ml or » 1 ng was used
as input to PCR.

Product	Size	Item No.
ProPrep™ Genomic 96/100	96-100 preps	PPG-100
ProPrep™ Genomic 960/1000	Up to 1000 preps	PPG-1000

ProPrep™ BAC Mini
Mini-Prep for BACs & Large Plasmids Using ProCipitate™

• Ideal for mini-prep BAC template preparation from 1.5-2.0 ml 2xYT cultures.
• Provides high-quality DNA suitable for automated sequencing.
• Utilizes ProCipitate™ strategy which minimizes shearing and improves yield.
• Adaptable to 96 well and automation.
• Protocols served the Human Genome Sequencing Project

ProPrep™ BAC Mini is a complete microfuge based purification system based upon the proprietary reagent,ProCipitate™.ProCipitate™ has been demonstrated to provide high quality DNA suitable for automated fluorescent sequencing of small to large insert DNA.

ProPrep™ BAC Mini starts with 2ml overnight cultures, and then utilizes ProCipitate™ in a modified alkaline lysis protocol. While this kit is adaptable to 96-well plates, plastic components are not stocked but recommendations for suitable suppliers can be made.

Product	Size	Item No.
ProPrep™ BAC Mini	96-100 preps	PMK-100
ProPrep™ BAC Mini	Up to 500 preps	PMK-500
ProPrep™ BAC Mini	Up to 1000 preps	PMK-1000

ProCipitate™ and related products can be customized to fit specific needs. It also can be supplied in bulk quantities. Please contact our sales office or any of our worldwide distributors for more information.

CONTACT:
Dr. Swapan Roy & Matthew Kuruc

Biotech Support Group LLC
1 Deer Park Drive, Suite M
Monmouth Junction NJ 08852
732-274-2866 Worldwide
800-935-0628 North America
sales@biotechsupportgroup.com
http://www.biotechsupportgroup.com

*For research use only

References

Patents

Composition and utility patents for ProCipitateä and related technologies are covered under U.S. Patents Numbers 5,294,681, 5,453,493 & 5,658,779.

U.S. Patent Number 5,538,870, Method for Preparing Nucleic Acids For Analysis And Kits Useful Therefore. This patent shows the beneficial effects of ProCipitate™ in protocols which neutralize SDS with non-ionic detergents, are PCR compatible, and require no alcohol precipitation.

Plasmids, Cosmids, BACs

Huang, Guyang Matthew, et al. “A high-throughput plasmid DNA preparation method.” Analytical biochemistry 223.1 (1994): 35-38.

Klein, Robert R., Daryl T. Morishige, Patricia E. Klein, Jianmin Dong, and John E. Mullet. “High throughput BAC DNA isolation for physical map construction of sorghum (Sorghum bicolor).” Plant Molecular Biology Reporter 16, no. 4 (1998): 351-364.

Kelley, Jenny M., et al. “High throughput direct end sequencing of BAC clones.” Nucleic acids research 27.6 (1999): 1539-1546.
Applied Biosystems User Bulletin. Subject: Sequencing Large DNA Templates.

Osoegawa, Kazutoyo, et al. “Bacterial artificial chromosome libraries for mouse sequencing and functional analysis.”Genome Research 10.1 (2000): 116-128.

Sonstegard, Tad S., et al. “Comparative map alignment of BTA27 and HSA4 and 8 to identify conserved segments of genome containing fat deposition QTL.” Mammalian Genome 11.8 (2000): 682-688.

Locke, John, et al. “A physical map of the polytenized region (101EF–102F) of chromosome 4 in Drosophila melanogaster.”Genetics 155.3 (2000): 1175-1183.

Reddy, O. Umesh K., Alan E. Pepper, Ibrokhim Abdurakhmonov, Sukumar Saha, Johnie N. Jenkins, Thomas Brooks, Yuksel Bolek, and Kamal M. El-Zik. “New dinucleotide and trinucleotide microsatellite marker resources for cotton genome research.”J Cotton Sci 5, no. 2 (2001): 103-113.

David C. Bruce; Mark O. Mundt; Kim K. McMurry; Linda J. Meincke; Donna L. Robinson; Norman A. Doggett; Larry L. Deaven. BAC Library End Sequencing in Support of Whole Genome Assemblies poster session. DOE Joint Genome Institute and Center for Human Genome Studies, Los Alamos National Laboratory

Quiniou, Sylvie MA, Takayuki Katagiri, Norman W. Miller, Melanie Wilson, William R. Wolters, and Geoffrey C. Waldbieser. “Construction and characterization of a BAC library from a gynogenetic channel catfish Ictalurus punctatus.” Genetics Selection Evolution 35, no. 6 (2003): 673-684.

Campbell, Barbara J., Jeffrey L. Stein, and S. Craig Cary. “Evidence of chemolithoautotrophy in the bacterial community associated with Alvinella pompejana, a hydrothermal vent polychaete.” Applied and environmental microbiology 69, no. 9 (2003): 5070-5078.

Chi, J. X., et al. “Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping.” Chromosoma 114.3 (2005): 167-172.

Cohen, Stephanie M., Terrence S. Furey, Norman A. Doggett, and David G. Kaufman. “Genome-wide sequence and functional analysis of early replicating DNA in normal human fibroblasts.” BMC genomics 7, no. 1 (2006): 301.

Alsop, Amber E., Andrew E. Teschendorff, and Paul AW Edwards. “Distribution of breakpoints on chromosome 18 in breast, colorectal, and pancreatic carcinoma cell lines.” Cancer genetics and cytogenetics 164.2 (2006): 97-109.

McDermott, Brian M., Yukako Asai, Jessica M. Baucom, Shraddha D. Jani, Yaneth Castellanos, Gustavo Gomez, James M. McClintock, Catherine J. Starr, and A. J. Hudspeth. “Transgenic labeling of hair cells in the zebrafish acousticolateralis system.”Gene Expression Patterns 10, no. 2 (2010): 113-118.

Enteric Viruses and Environmental Sampling

Schwab KJ, De Leon R, Sobsey MD “Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR.” Applied and Environmental Microbiology 61.2 (1995): 531-537.

Schwab KJ; De Leon; Sobsey MD. “Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR.” Applied and Environmental Microbiology 62.6 (1996): 2086-2094.

LA Jaykus, Ricardo De Leon and MD Sobsey. “A virion concentration method for detection of human enteric viruses in oysters by PCR and oligoprobe hybridization.” Applied and Environmental Microbiology 62.6 (1996): 2074-2080.

Rodríguez, Roberto A., Lauren Thie, Christopher D. Gibbons, and Mark D. Sobsey. “Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters.” Journal of virological methods 181, no. 1 (2012): 43-50.

Richards, Gary P., and Gail E. Greening. “Detection of enteric viruses in shellfish.” Molluscan Shellfish Safety. Springer Netherlands, 2014. 177-183.

Infectious Disease and Pathogen Detection, Paraffin-embedded Tissues

Gyimesi, Zoltan S., et al. “Detection of Mycobacterium avium subspecies avium in formalin-fixed, paraffin-embedded tissues of captive exotic birds using polymerase chain reaction.” Journal of Zoo and Wildlife Medicine(1999): 348-353.

Miller, Janice M., Allen L. Jenny, and Jay L. Ellingson. “Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues.” Journal of Veterinary Diagnostic Investigation 11.5 (1999): 436-440.

Thornton, Charles G., Kerry M. MacLellan, Judith R. Stabel, Christine Carothers, Robert H. Whitlock, and Selvin Passen. “Application of the C18-carboxypropylbetaine specimen processing method to recovery of Mycobacterium avium subsp. paratuberculosis from ruminant tissue specimens.” Journal of clinical microbiology 40, no. 5 (2002): 1783-1790.

J.L.E. Ellingsona,J.R. Stabela, R.P. Radcliff b, R.H. Whitlockc, J.M. Miller. Detection of Mycobacterium avium subspecies paratuberculosisin free-ranging bison (Bison bison) by PCR. Molecular and Cellular Probes 19 (2005) 219–225.

Enzyme Removal (DNA)

Kozekov, Ivan D., Robert J. Turesky, Guillermo R. Alas, Constance M. Harris, Thomas M. Harris, and Carmelo J. Rizzo. “Formation of deoxyguanosine cross-links from calf thymus DNA treated with acrolein and 4-hydroxy-2-nonenal.” Chemical research in toxicology 23, no. 11 (2010): 1701-1713.

Blood (DNA) PCR

Krupey, John, Swapan Roy, and Bhavin Desai. “100,000+ PCRs Possible from 10 ml Blood.”

Protein Enrichment

Burton, Jack D., Richard N. Bamford, Christian Peters, Angus J. Grant, Gloria Kurys, Carolyn K. Goldman, Jennifer Brennan, Erich Roessler, and Thomas A. Waldmann. “A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.” Proceedings of the National Academy of Sciences 91, no. 11 (1994): 4935-4939.