Remote Molecular Biology: Difference between revisions
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==[[PCR-based Methods]]== | ==[[PCR-based Methods]]== | ||
==[[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®ion=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®ion=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. | |||
==[[Culture-based Methods]]== | ==[[Culture-based Methods]]== | ||
Revision as of 23:11, 8 April 2019
Topics to Investigate
Articles / Papers to read
Relevant Pathogens
General Lab Methods
PCR-based Methods
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®ion=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®ion=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.