5-Fluoroorotic Acid Monohydrate (FOA, 5-FOA)

Essential information

CAS:207291-81-4

MW: 192.1

Formula: C5H3FN2O4.H2O

Nomenclature:5-Fluoro-4-Pyrimidinecarboxlic Acid; 5-Fluoro-1,2,3,6-Tetrahydro-2,6-Dioxo-(9CI); 1,2,3,6-Tetrahydro-2,6-Dioxo-5-Fluoro-4-Pyrimidinecarboxlic Acid.

Solubility

    We recommend to first partially solubilize FOA in ddH2O, then autoclave for complete solubility

    NH4OH/H2O (1:1): 50mg/ml

    DMSO: Very soluble in DMSO. High concentrations of DMSO are toxic to cells. Low concentrations are recommended.

    H2O: Slightly soluble. in H2O. Readily solubilized with heat. Withstands boiling.

    Ethanol: Slightly soluble in ethanol.

    Methanol: Slightly soluble in methanol.

Appearence:Off-white to light yellow powder.

Melting Point:~258°C FOA is stable at high heat (i.e. boiling and autoclave conditions.) Refer to United States Biological reference article on stability testing data

Storage and Stability:

    Lyophilized powder may be stored at RT or 4°C. Refer to Solubility. Protect reconstituted product from light and store at 4°C during use. For long-term storage, aliquot and store at -20°C. Aliquots are stable for 6 months at -20°C.

Thermostability of 5-Fluoroorotic Acid (FOA)

    Researchers using the budding yeast, Saccharomyces cerevisiae, have a great number of genetic tools at their disposal. Among these are the plasmids of many types and copy number that have been developed for use in S. cerevisiae. The selection for transformants with these plasmids often relies on the complementation of a genomic mutation in the genes required to synthesize various amino acids (leucine, histidine, tryptophan, etc.) or nucleotide bases (generally adenine or uracil). Recently the use of aurine resistance has been utilized.

    Most laboratory strains lack a functional URA3 gene and can be complemented to uracil auxotrophy by supplying URA3 on a plasmid or by integration into the genome. URA3 strains can also be selected against by including 5-Fluoroorotic Acid (5-FOA) in the growth media. Cells with wild-type URA5 and URA3 genes convert the 5-FOA into the toxic substance 5’ Fluorouridine monophosphate, severely limiting growth of the cell. The ability to carry out positive and negative selection with one marker has allowed yeast researchers to devise ingenious screens and genetic schemes.

    The use of 5-FOA is not always as simple as theory might dictate. Many researchers have a difficult time getting reproducible results from experiments using 5-FOA. The reasons for this are not always apparent. The anecdotes have lead to researchers to question the stability of 5-FOA in light, heat, solution, etc. Some protocols even recommend boiling to completely dissolve 5-FOA.

    The following experiment was designed to demonstrate the effect of these variable conditions on the end use of 5-FOA, growth inhibition of uracil auxotrophs in the laboratory.

    5-FOA was obtained from US Biological frozen stocks and aliquots incubated at -20° (control) or 45° C for 16 hours (to simulate an unprotected ground shipment in an overheated UPS truck). To investigate the effects of boiling, we also added 5ml of water to an aliquot of 5-FOA and incubated in boiling water for 10 minutes. We then added the 5-FOA at 1g/liter to synthetic complete media (26.7 g/liter Drop-out Base, 2g/liter SC Drop-out Amino Acid Supplement, stirred until no particulate matter could be observed (generally 10-30 minutes). The media was filtered through a 0.2um filter to sterilize.

    We inoculated the 5-FOA media with an overnight culture of W303a pRS316 grown in SC-ura (26.7g/liter Drop-out Base, 2g/liter Drop-out mix minus uracil. Growth was followed by measuring the OD600.

    All 5-FOA samples, regardless of the thermal abuse heaped upon them, severely limited the growth of the URA3 culture relative to the SC control. In fact, the boiled sample consistently performed better than the non-boiled control. This might be due to the complete solublization of the 5-FOA.

    We conclude that 5-FOA is very thermotolerant for at least short periods of 1-2 weeks. It can be safely shipped via ground without temperature protection.

Procedure

1.All materials, reagents, glassware, tips, etc. must be autoclaved or certified sterile by the manufacturer prior to use at any point in this procedure.

2.All relevant data and results will be recorded on DOC NO: 200–009, Request for QC Testing. Include lot numbers of all materials used, genus, species, and ATCC accession number of test organism, incubator number and environmental conditions in incubator.

3.The preferred fungal cell line for this test is Candida albicans strain CFD1.

4.Prepare SC agar plates and URA Drop-out plates two days prior to scheduled testing.

5.Inoculate 5ml Ura Drop-out media with a colony of CFD1. Incubate overnight at 30°C.

6.Prepare SC by combining 1.335g Drop-out Base and.1g SC Drop-out Mix in 50ml final volume and filter sterilize. Prepare SC-FOA by combining 1.335g Drop-out Base,.1g SC Drop-out Mix, and.050g 5'FOA in 50ml total volume and filter sterilize. Avoid exposing 5'FOA to light by weighing the FOA in the dark and keeping the solutions in foil covered 250ml flasks.

7.Add 1ml of overnight culture to the SC and SC-FOA flasks. Incubate overnight at 30°C with shaking. Dilute each culture 1:100 and 1:10,000.

8.Dilute each culture 1:100 and 1:10,000.

9.Plate 100ul of the original culture and each dilution on two plates each of URA Drop-out and SC Drop-out.

10.Incubate 2days at 30°C.

11.Count the colonies on each plate containing 5–500 colonies. Calculate the density in the original culture.

12.Criterion for acceptance of 5' FOA: There must be >1000X as many colonies on the SC Drop-out plate as the URA Drop-out plate at any given dilution.

FAQ

1.What is the pH of selection medium?

A:FOA selection breaks down above pH 4.5. This problem is consistent with only the protonated form of FOA permeating cells. It turns out that the standard recipe for FOA plates is pH 2.8. Some of this acidity is due to the FOA, which you have less of, so your plates must be at slightly higher pH. - 5-FOA has no effect at all at pH 6 or 6.2, pretty good at pH 5.4, and full effect at 4 and below. This is as 2X filtered medium at R.T., before mixing with 4% hot agar. After solidifying, the plates have a slightly higher pH, as measured with a pH stick/paper..

2.How long can prepared plates with 5-FOA be stored?

A:Most laboratories store 5-FOA prepared plates for several months (3-6 months). The longest storage time that I found was for 9 months, and for >12 months Store at 4°C, without drying out (wrapped in parafilm).

3.How much 5-FOA should I use?

A:FOA (1mg/ml) has been used as a selective agent in yeast molecular genetics; in the selection of Ura cells in a population of Ura+ cells; and in the selection of orotidine-5’-phosphate decarboxylase (OMPdecase) mutants of Saccharomyces cerevisiae.

FOA (0.1mg/ml) has been used in the positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius.

References

Kondo, S. et al., J. Bacteriology, 173, 7698, 1991.

Vidal, M. et al., Proc. Natl. Acad. Sci. USA, 93, 10321, 1996.

Winston, F. et al., Genetics, 107, 179, 1984.

Boeke, J.D. et al., Mol. Gen. Genet., 197, 345, 1984.

To prepare 5-FOA, some researchers have dissolved the material at about 50 mg/mL in 4 M ammonium hydroxide, producing a clear solution, with sonication or application of heat as needed.