Remote Molecular Biology

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Relevant Pathogens

General Lab Methods

PCR-based Methods

Fluorescent Tagging of Primers

Fluorescent tagging has historically been used to label proteins. As such, there are cheap commercial means to obtain fluorescently tagged proteins and also efficient ways of tagging proteins in the lab.[1–3] However, primers can also be fluorescently tagged for use in PCR. There are a range of tags to be used depending on the desired color.

    1.6-FAM = blue; the most intense dye 
    2.HEX = green 
    3.NED = yellow; replacement for TET/TAMRA 
    4.ROX = red; used as a size standard label 
    5.TAMRA = red or yellow; can be an alternative size standard label 
    6.TET = yellow; least intense dye and has strong overlap with 6-FAM 

Despite the range of options, ordering fluorescently tagged primers can be expensive. For example, ordering them from Integrated DNA Technologies will cost upwards of $80 per primer. [4] The price is even more egregious from places like Sigma Aldrich. Furthermore, if one wanted to set up a molecular beacon by tagging one end with a primer and the alternate end with a quencher, that would cost even more. For example, ordering an oligonucleotide with the dark quencher, Dabcyl, at the 3’ end costs $80 at the cheapest. It is then easy to imagine how expensive ordering an oligonucleotide with both a 5’ fluorophore and a 3’ quencher.

The alternative is to add a fluorescent tag ourselves after ordering the primers.[5] This has been historically done by first adding phosphoramidite and then adding a dye-NHS ester. However, companies like ThermoFisher now sell all-in-one reagents that complete the tagging reaction in one step. However, these are likely to cost upwards of $300.[6] The cost is not far off from the traditional method of purchasing the two reagents separately.

Given these prices, I think it is more financially advantageous to explore dye-based qPCR methods.[7] In these methods, a dsDNA binding dye such as SYBR Green is used. “The dye displays weak background fluorescence that increases dramatically upon binding to dsDNA. Thus, amplification of the target sequence results in an increase of fluorescence that is directly proportional to the amount of dsDNA present at each PCR cycle. This type of qPCR assay requires only two sequence-specific primers, making it a rapid and cost-effective way to interrogate a large number of samples/targets.”

One disadvantage of intercalating dye-based methods is that they detect any dsDNA produced in the reaction. This includes off target amplification products and primer-dimers, which results in inaccurate quantification.” One way to verify the results would be to perform a denaturation curve to verify reaction specificity.


1. Galbán J, Andreu Y, Sierra JF, Marcos S de, Castillo JR (2001) Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review. Luminescence, 16(2):199–210. https://doi.org/10.1002/bio.633

2. Liu H-W, Chen L, Xu C, Li Z, Zhang H, Zhang X-B, Tan W (2018) Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging. Chemical Society Reviews, 47(18):7140–7180. https://doi.org/10.1039/C7CS00862G

3. Toseland CP (2013) Fluorescent labeling and modification of proteins. Journal of Chemical Biology, 6(3):85–95. https://doi.org/10.1007/s12154-013-0094-5

4. Fluorophores. https://www.idtdna.com/site/Catalog/Modifications/Category/3

5. Giusti WG, Adriano T (1993) Synthesis and characterization of 5’-fluorescent-dye-labeled oligonucleotides. Genome Research, 2(3):223–227. https://doi.org/10.1101/gr.2.3.223

6. Oligo Synthesis Reagents. https://www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catDisplayStyle&catKey=601610&filterDispName=Oligo%2BSynthesis%2BReagents&_bcs_=H4sIAAAAAAAAAMWQTWvDMAyGf40vMytO3K25lpaW0TLGwnbXbCU2%2BKPYzkL%2B%2FZSNsB1KrwOh15KF%0A%2FLy%2Br5hoXlLUgyqZs%2FqRt5g%2BrcJ8o29KuWQmt6w%2BUIzjuCoGk4%2BdzaQrFT21h0wJAyUTPZLEpOnS%0AFO9oBavlHKIpacC5FhtBUomKJh%2BqtajuqDzDB39F6DEQBATNdwa9VeAWitsQctuI5WFSBQVc7Bes%0At5YSaJtQFTq5wuRBec3k%2Fun9%2BEzDe5svDqYdFOxjmugTqHnCiSa%2BCf%2Fa6Ijpuo9ZN%2FWPnyMGTOB%2B%0AbfAuJn62HfJWWQwK%2F9vXTHrN1xd36rmWKAIAAA%3D%3D

7. Dye-based qPCR & RT-qPCR | NEB. https://www.neb.com/applications/dna-amplification-pcr-and-qpcr/qpcr-and-rt-qpcr/dye-based-qpcr

Magnetic Beads for DNA isolation

How magnetic beads work [1]

Early magnetic beads consisted of an iron-oxide core coated with silane. The surface of these beads was bound with molecules containing a free carboxylic acid. This functional group bound to DNA or RNA. Environmental salt concentration was varied to control the strength of the bonds between functional groups and nucleic acid. This allowed for controlled reversible binding. Different functional groups such as -COOH, -OH or -NH2 are used. For specific compounds some beads are functionalize with immobilized affinity ligands, hydrophobic ligands, or ion-exchange groups.

Recyclable magnetic beads [2]

Recyclable magnetic beads fall into 3 categories. core–shell structure particles, matrix-dispersed structure particles and hollow structure particles. These are regarding the structure of the beads. Each bead can be recycled in various ways. These methods could be classified into four types: direct reuse, washing treatment, chemical treatment and high-temperature calcination.

     1.“Direct reuse means that recyclable magnetic particles are reused directly after magnetic separation without extra processing. This method is suitable for applications in which the remnants do not affect the next cycle… this includes enzyme immobilization and pathogenic bacterial inactivation.”
     2.Washing treatment shows a wider range of applications, such as enzyme immobilization, bacterial immobilization, protein extraction, and DNA adsorption. However, washing treatments may affect binding ability 
     3.Example for a desorption experiment: “NaOH solution was added to weaken the electrostatic interaction between the polyaniline chains and dsDNA. Afterward, the recovered particles were washed with deionized water and HCl, bringing their fully active form (positively charged) to the next adsorption–desorption cycle. The adsorption efficiency was 90% after three cycles. However, acid and base solutions may destroy the secondary structure and affect the subsequent use of DNA.”
     4.High-temperature calcination also presents the risk of denaturing the secondary structure of the beads. Typically used when the other three methods fail. 

(1) Safarik, I.; Safarikova, M. Magnetic Techniques for the Isolation and Purification of Proteins and Peptides. Biomagn Res Technol 2004, 2, 7. https://doi.org/10.1186/1477-044X-2-7.

(2) Liu, Z.; Liu, Y.; Shen, S.; Wu, D. Progress of Recyclable Magnetic Particles for Biomedical Applications. J. Mater. Chem. B 2018, 6 (3), 366–380. https://doi.org/10.1039/C7TB02941A.

Freezing PCR reagents

Pre-made PCR beads

From detail given from: 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).

PCR beads are 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, these products are rather expensive with 480 individual pellets, which is equivalent to 480 possible reactions costing $846.00 from GE healthcare. This is consistent among all retailers as Sigma Aldrich sells a similar product for $816.00. This comes to a cost of ca. $2 per reaction.

Selfmade Setups

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

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.

Culture-based Methods

Sequencing Methods

Bioinformatics Methods

Inventory/Prices