Producing Nucleotides for PCR

Background

Nucleotides are notoriously unstable unless stored at the correct temperatures. However, one alternative may be to produce them onsite. This can be also prove to be financially advantageous as the price of dNTP is $120 for 4.9 mg or $24.50 per mg (from Thermofisher).

Synthesis methods

From: 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.

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.

Procedure

1. nucleotidyl kinase with acetokinase was isolated from the E. coli MRE-600 cells.

2. DNA was prepared from salmon milt (final purity: 80%)

3. S1 Nuclease was immobilized using the method developed by Khomov (note that the source "Khomov, V. V., Sizov, A. A., Masycheva, V. I., and Zagrebelnyi, S. N. (1997) Biotekhnologiya, 3,29,34" cannot be found

4. 100 ml of DNA solution was supplemented with 40 ml of DNase solution in 20 mM sodium acetate buffer containing 5 mM MgSO4 and incubated at 37°C for 1.5 h. The reaction was stopped by addition to the reaction mixture of 1 M cooled HClO4 at the ratio of 1 : 1

5. Resulting oligonucleotides were hydrolyzed further by adding ZnSO4 until it reached a 1mM concentration and then passing the mixture through a 100 ml column with immobilized S1 nuclease equipped with a thermostatted jacket, at the rate of 100 ml/h at 44°C. The reaction was stopped with 1 M HClO4 added at the ratio 1 : 1.

6. Individual dNMP and NMP were separated by chromatography on anion exchanger Dowex 1×2 at acidic pH (0.003 M HCl) in a linear gradient of NaCl (0.1-0.4 M).

       Using a crude estimation of cost
       1. $60; S1 nuclease (10,000 units) 
       2. $80; DNase from bovine pancrease (10 mg) 
       3. $45; ATP (0.25 mL at 100 mM or 12.6 mg) 
       4. $140; Lithium potassium acetyl phosphate (500 mg)
       5. N/A; nucleotidyl kinase and acetokinase was isolated from the E. coli MRE600 cells

From 10 grams of dNMP, they had different yields for each nucleotide: from 1.44 grams for CTP to 3.15 for GTP). There is also a discrepancy between the stoichiometric ratios used for each nucleotide. The largest ratios seemed to be 150 mg dGMP:1.96 mg ATP:32.5 mg lithium acetylphosphate. This step is relatively cheap, only costing about $20. The article fails to mention how much DNase and S1 nuclease was used. However, even using a cautious estimate of $100 for the production of 10 grams of usable DNA results in a final cost of 1 cent per mg. This is much cheaper than ordering the nucleotides online. Even considering the 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, the financial advantageous cannot be ignored. This is especially true when considering that the cost of procedural training and protein isolation is only required once at the beginning of the experiment.

The research is backed by a second source 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

Increasing procedural efficiency and decreasing the cost

1. Perform a one pot synthesis (note that the immobilization of S1 nuclease was made to increase the efficiency of the protein. Thus we can theoretically compensate by increasing the amount of protein used). However, no research has been done on this.

2. Skip the chromatography step - hypothetically, if we add nucleotides in excess, a slightly shifted ratio won't affect the functionality of the PCR

3. Increase the efficiency of our added ATP by incorporating enzyme-catalyzed ATP recycling (Alissandratos, A.; Caron, K.; Loan, T. D.; Hennessy, J. E.; Easton, C. J. ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR. ACS Chem. Biol. 2016, 11 (12), 3289–3293. https://doi.org/10.1021/acschembio.6b00838.) However, this procedure necessitates the addition of several enzymes and reagents that may need to be removed lest they interfere with the polymerase. This can potentially be solved through chromatography.