Remote Molecular Biology

Freezing PCR reagents to increase shelf life

TBA

(1) Klatser, P. R.; Kuijper, S.; van Ingen, C. W.; Kolk, A. H. J. Stabilized, Freeze-Dried PCR Mix for Detection of Mycobacteria. J Clin Microbiol 1998, 36 (6), 1798–1800.

(2) In Pursuit of a Shelf-Stable qPCR Mix https://opsdiagnostics.com/notes/LyoqPCRreagents.html (accessed Apr 9, 2019).

(3) Seise, B.; Pollok, S.; Seyboldt, C.; Weber, K. Dry-Reagent-Based PCR as a Novel Tool for the Rapid Detection of Clostridium Spp. Journal of Medical Microbiology 2013, 62 (10), 1588–1591. https://doi.org/10.1099/jmm.0.060061-0.

PCR beads

PCR beads are typically pellets of pre-formulated PCR regents that have been freeze-dried to increase shelf life. They generally contain Taq DNA polymerase, nucleotides, BSA and sometimes stabilizers or metal cofactors. As such, the only thing that needs to be added are water, primers and template DNA. This not only affords consistent PCRs time and time again, but also minimizes pipette tip usage as they are typically packaged in 96 well plates.

However, they are quite pricy with 480 individual pellets (and thus 480 possible reactions) costing $846.00 from GE healthcare(https://www.fishersci.com/shop/products/ge-healthcare-illustra-puretaq-ready-to-go-pcr-beads-4/p-4007188). This is a rather consistent pricing as Sigma Aldrich sells a similar product for $816.00 (https://www.sigmaaldrich.com/catalog/product/sigma/ge27955702?lang=en&region=US)

Making the Polymerase Chain Reaction Easier with PCR EdvoBeads™ http://www.edvotek.com/Making-the-Polymerase-Chain-Reaction-Easier-with-PCR-EdvoBeads (accessed Apr 9, 2019).

Producing Nucleotides

Nucleotides are notoriously unstable unless stored at the correct temperatures. However, one alternative may be to produce them onsite. A traditional way of doing such is to use pancreatic DNase from Serratia marcescens and phosphodiesterase from venom. However, this is economically unfeasible as the price of venom has reached $6000 per gram. An alternative for DNA hydrolysis is to use S1 Nuclease from commercially available A. oryzae and DNAse from cattle pancreases. This hydrolysis reaction can also catalyzed by the addition of ZnSO4. Phosphorylation of the subsequent dNMPs is performed by adding ATP, lithium acetylphosphate and an assortment of kinases.

1. Bochkov, D. V.; Khomov, V. V.; Tolstikova, T. G. Hydrolytic Approach for Production of Deoxyribonucleoside-and Ribonucleoside-5′-Monophosphates and Enzymatic Synthesis of Their Polyphosphates. Biochemistry (Moscow) 2006, 71 (1), 79–83. https://doi.org/10.1134/S0006297906010123.

2. Bao, J.; Ryu, D. D. Y. Total Biosynthesis of Deoxynucleoside Triphosphates Using Deoxynucleoside Monophosphate Kinases for PCR Application. Biotechnology and Bioengineering 2007, 98 (1), 1–11. https://doi.org/10.1002/bit.21498.

Using a crude estimation of cost

1. $60; S1 nuclease (10,000 units) https://www.thermofisher.com/order/catalog/product/EN0321

2. $80; DNase from bovine pancrease (10 mg) https://www.sigmaaldrich.com/catalog/product/sigma/dn25?lang=en&region=US

3. N/A; nucleotidyl kinase and acetokinase was isolated from the E. coli MRE600 cells

4. $45; ATP (0.25 mL at 100 mM or 12.6 mg) https://www.thermofisher.com/order/catalog/product/R0441

5. $140; Lithium potassium acetyl phosphate (500 mg) https://www.sigmaaldrich.com/catalog/product/sigma/a0262?lang=en&region=US

From 10 grams of dNMP they yielded a range from 1.44 (for CTP) to 3.15 (for GTP) DNTP. There also seems to be a discrepancy between the stoichiometric ratios used for each nucleotide. The largest ratios seemed to be 150 mg dGMP, 1.96 mg ATP, and 32.5 mg lithium acetylphosphate. This means that this step is relatively cheap, only costing about $20. The article fails to mention how much DNase and S1 nuclease was used. However, an extensive amount of lab work was done to purify the DNA sample and also immobilize the S1 nuclease in a column of aminobutyl-(AB)-Bio-Gel P-2. This includes the usage of several buffers and inorganic salts that act as catalysts. We use a cautious estimate of $100 for the production of 10 grams of usable DNA.

This can be compared to the price of dNTP which is $120 for 4.9 grams (https://www.thermofisher.com/order/catalog/product/R0191). When considering the work necessary for the the production of dNTP, it seems purchasing of dNTP is much more favorable.