Remote Molecular Biology

Freezing PCR reagents to increase shelf life

There are several methods that they use to increase the shelf life of PCR reagents

From: 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. PCR mix was first created using a solution of buffer, primers, DNA polymerase, deoxynucleoside triphosphates dATP, dCTP, dGTP, and dTTP, and UDG. Trehalose was added to the PCR mix at 5% wt/vol (the tested optimal proportion). Lyophilization was performed on batches of 15 reactions at a time in a Klee pilot freeze-dryer. The freeze-dried mixtures were reconstituted to their original volume with distilled water.

     Some notes on lyophilization (based of the usage for bacteria)
     1. Bacteria need a lyoprotectant which helps them survive the freeze drying process, such as 10% skim milk. The             
     lyoprotectant  stabilizes the cells when water is removed and allows the sample to retain its shape 
     during and after processing.  Disaccharides such as sucrose and trehalose are excellent lyoprotectants.
     Freeze Drying Process
     2. Freeze drying can be divided into three stages: freezing, primary drying, and secondary drying. 
     Freezing: often done under vacuum so that water can be pulled from ice into the headspace. Samples are dried 
     afterwards to remove residual moisture
     Primary drying: removes readily available frozen water.  
     Secondary drying: forces out residual water by increasing the temperature of the sample.  
     Freeze dried proteins can be stored at relatively warm temperatures as long as no moisture gets to the sample. 

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

Reaction solutions were prepared in 200 µl eight tube PCR strips. Each tube contained 19 µl of qPCR mix which was taken from a master mix of: 1. 100 µl 2X Lyophilization Reagent 2. 10 µl primer mix (20X concentrate) 3. 1 µl Taq DNA polymerase 4. 4 µl dNTPs at 25 mM each nucleotide The mixes were flash frozen in a 96 well Cooling Block, which was pre-chilled to -80°C in a laboratory freezer. The blocks were transferred to a freeze-dryer that was pre-chilled to -40°C and the tubes were equilibrated to -40°C for 1 hr, followed by the application of vacuum at 200 mtorr. The temperature was then adjusted to -15°C for primary drying. After 4 hours, the temperature was increased to 20°C for secondary drying for 45 minutes. The tubes were then immediately capped and vacuum sealed in Mylar pouches at 4°C. The pellets were rehydrated in 19 µl 1X PCR buffer (containing 5 mM MgCl2).

Some procedures also don't use a lyophilizing agent. From: 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. Mixtures of BSA, PCR buffer, MgCl2, primers and dNTPs were applied to polyolefin foils (The polyolefins polypropylene (PP) and PE are supplied by Nowofol or Analytik Jena) and dried for 2 h at room temperature under a fume hood. The dried spots were cut and stored at ambient temperatures in a 200 ml reaction tube. The dried reagents of polyolefin foils were reconstituted by adding nuclease-free deionized water

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.

1. 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).

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)

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 mg (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.