Short Technical Reports hybridization amount to 10 µg reduced the detectable transcript number by 8%. In summary, the data we have shown in this report demonstrate the successful hybridization to Affymetrix DNA chips using LCM-generated cRNA after two rounds of T7 linear amplification. About 30% of the approximately 7000 gene transcripts queried were detected in the oral cancer tissue. This compared favorably with the number of transcripts detected in similar experiments using comparably sized samples from other types of cells harvested by traditional methods (5). REFERENCES 1.Emmert-Buck, M.R., R.F. Bonner, P.D. Smith, R.F. Chuaqui, Z. Zhuang, S.R. Goldstein, R.A. Weiss and L.A. Liotta. 1996. Laser capture microdissection. Science 274:998-1001. 2.Katcharmina, J.E., P.B. Crino and J. Eberwine. 1999. Preparation of cDNA from single cells and subcellular regions. Methods Enzymol. 303:3-18. 3.Lockhart, D.J., H. Dong, M.C. Byrne, M.T. Follettie, M.V. Gallo, M.S. Chee, M. Mittmann, C. Wang et al. 1996. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 14:1675-1680. 4.Luo, L., R.C. Salunga, H. Guo, A. Bittner, K.C. Joy, J.E. Galindo, H. Xioa, K.E. Rogers et al. 1999. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat. Med. 5:117-122. 5.Mahdevapa, M. and J.A. Warrington. 1999. A high-density probe array sample preparation method using 10- to 100-fold fewer cells. Nat. Biotechnol. 17:1134-1136. 6.Saizieu, A., U. Certa, J. Warrington, C. Gray, W. Keck and J. Mous. 1998. Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nat. Biotechnol. 16:45-48. 7.Simone, N.L., R.F. Bonner, J.W. Gillespie, M.R. Emmert-Buck and L.A. Liotta. 1998. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet. 14:272-276. 8.Suarez-Quian, C.A., S.R. Goldstein, T. Pohida, P.D. Smith, J.I. Peterson, E. Wellner, M. Ghany and R.F. Bonner. 1999. Laser capture microdissection of single cells from complex tissues. BioTechniques 26:328-335. 9.Wodicka, L., H. Dong, M. Mittman, M.H. Ho and D.J. Lockhart. 1997. Genome-wide expression monitoring in Saccharomyces cerevesiae. Nat. Biotechnol. 15:1359-1367.

(NIDCR) grant nos. R29 DE11983 (to R.T.), P01 DE12467 (to D.T.W.W.) and P30 DE11814 (to D.T.W.W.), Harvard University William F. Milton Fund (to R.T.) China Site Key Basic Research Program grant no. G199805123 (to X.Z.) and Oral and Maxillofacial Surgery Foundation Research Support Grant (to H.O.) in collaboration with Janet A. Warrington and Mamatha Mahadevappa of the Affymetrix Corporation, Santa Clara, CA. H.O. is a research fellow of the Japanese Society for the Promotion of Science. Address correspondence to Dr. Randy Todd, Massachusetts General Hospital, Department of Oral and Maxillofacial Surgery, Warren 1201, 1 Fruit Street, Boston, MA 02114, USA. e-mail: randy_ [email protected] Received 15 March 2000; accepted 15 May 2000.

H. Ohyama, X. Zhang1, Y. Kohno, I. Alevizos, M. Posner2, D.T. Wong and R. Todd3 Harvard School of Dental Medicine Boston, MA, USA 1China Medical University Shenyang, China 2Dana-Farber Cancer Institute Boston, MA, USA 3Massachusetts General Hospital Boston, MA, USA

Extended Stability of Restriction Enzymes at Ambient Temperatures BioTechniques 29:536-542 (September 2000)

ABSTRACT H.O. and X.Z. contributed equally and should both be considered as first author. This work was supported by the National Institute of Dental and Craniofacial Research 536 BioTechniques

The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduc-

tion in activity was observed for three enzymes (HindIII, EcoRI and Tsp509I) held at ambient temperature or 4°C for the period of study (12 months), while activity was observed for up to 12 weeks after storage at 37°C, which was considerably better than following desiccation with trehalose, a recognized preservation technique. A larger trial of 23 different restriction enzymes held at room temperature for one week showed that all enzymes retained significant activity. As a practical demonstration of the usefulness of this finding, enzymes were posted to Africa by conventional mail (cost $1 US) and shown to retain activity upon arrival after three weeks in transit (compared to a cost of $1000 US by cold-chain transportation). Supplying enzymes to third-world markets should now be possible by removing the necessity for cold-chain transport. After arrival, enzymes can simply be stored in a standard domestic refrigerator.

INTRODUCTION The importance of DNA restriction enzymes in recombinant DNA technology is unquestionable, with widespread applications in many diagnostic procedures such as restriction fragment length polymorphism (RFLP) and physical DNA mapping, in addition to their use in the cloning and modification of genes. Transport and storage are usually carried out at sub-zero temperatures, limiting the availability of these enzymes in countries with a less developed infrastructure. Many preservation techniques have been reported to improve the thermostability of enzymes, including conjugation to Sepharose and desiccation in the presence of stabilizing sugars such as trehalose. Lee et al. (2) found that B a mHI and EcoRI were stable for six months at 4°C when coupled to Sepharose, while Uritani et al. (4) reported stability of these enzymes for seven days at room temperature and up to 20 days at 4°C following desiccation with trehalose. Similar findings have been reported by Colaço et al. (1) and Rossi et al. (3) using trehalose as a desiprotectant. Methods such as these can be effective, but they greatly increase cost, and not all restriction enzymes are available in these modified forms. Also, storage at -20°C is still recommended Vol. 29, No. 3 (2000)

Short Technical Reports following reconstitution (Life Technologies product literature; Rockville, MD, USA). Surprisingly, no one appears to have examined the stability of restriction enzymes as supplied by manufacturers. In all publications concerning the protective effects of trehalose, enzymes desiccated without trehalose were used as controls, rather than untreated restriction enzymes (1–4). Our studies suggest that restriction enzymes may in fact be intrinsically quite stable as provided, sometimes exhibiting activity after several weeks of storage at 37°C or after one year at ambient temperature. If representative, these findings may increase the availability of restriction enzymes to more isolated regions.

MATERIALS AND METHODS

Medium-Term Storage

Long-Term Storage

A selection of 23 enzymes (Figure 2) from a variety of manufacturers were stored at -20ºC and ambient temperature for one week before the enzyme activity was tested.

Five enzymes chosen on the basis of availability in the laboratory were examined over the course of 12 months. These were HindIII, NarI, EcoRI (all 10 U/µL; Roche Molecular Biochemicals, Indianapolis, IN, USA), Tsp509I (5 U/µL; New England Biolabs, Beverly, MA, USA) and SmaI (10 U/µL; Promega, Madison, WI, USA). Aliquots (20 µL) were stored at -20°C, 4°C, ambient temperature (16°C–23°C) and 37°C until use. Enzyme activity was tested after 24 h, one week, two weeks, four weeks, eight weeks, 12 weeks, six months and one year.

Ambient Transport EcoRI, HindIII and SmaI were sent by conventional airmail in a plain envelope from Edinburgh, UK, to Boston, MA, USA and returned via the same route (total transit time six days) before activity was tested. Similarly, samples of HindIII and PstI were sent to Tanzania, Africa by conventional airmail (total transit time 21 days) and assayed upon arrival. Unless otherwise stated, each restriction digest contained 250 ng λ DNA (Promega) as substrate, 1 µL enzyme and 1 µL 0.1 mg/mL acetylated BSA (Promega) in a final volume of 10 µL. The buffers and reaction conditions varied according to the manufacturers’ recommendations. Reactions were left for 5 min, 1 h or overnight depending on the experiment. Digests were run on 1% agarose gels using TBE buffer (0.045 M Tris-Borate, pH 8.0, 0.001 M EDTA) on an Electro-Fast® gel system (ABgene, Epsom, UK). RESULTS AND DISCUSSIONS Long-Term Storage

Figure 1. Ethidium bromide-stained 1% agarose gels showing digestion of 250 ng λ DNA using a selection of enzymes after storage at 37°C, ambient temperature, 4°C and -20°C at various times for up to one year. Digestion was performed for 5 min and 1 h to roughly quantify the relative enzyme activity, with 1 µL enzyme used. Undigested λ DNA was run as a positive control. 538 BioTechniques

For a more sensitive assessment of enzyme activity, restriction digests were terminated after only a 5-min incubation and a 1-h incubation. Results are shown in Figure 1. For EcoRI, no appreciable decrease in activity was noted until eight weeks after transfer to 37°C, and some activity still remained after 12 weeks (the absence of activity of the 4°C sample after two weeks is most likely due to experimental error because subsequent reactions with the same enzyme exhibited activity). HindIII appeared to be even more stable, and it was not until six months after transfer to 37°C that a reduction in activity could be seen. For SmaI, although no activity was noted after storage for one week at 37°C, this is again likely to be due to experimental error Vol. 29, No. 3 (2000)

Short Technical Reports because a similar result was observed with the -20°C sample for digestion at this time point. Reduced activity was subsequently noted after two and four weeks of incubation at 37°C, although none remained after eight weeks. Since SmaI is generally considered a thermolabile enzyme with an incubation temperature of 25°C or 30°C, even this degree of thermostability is surprising. XbaI cuts λ DNA once, but using our electrophoresis protocol, we were unable to differentiate digested from undigested DNA. Therefore, an assessment of thermostability cannot be made for this enzyme from these results. For Tsp509I, full activity was noted after

six months at 37°C, although a considerable loss of activity was apparent after storage for one year at this temperature. With the exception of SmaI (stable to at least 12 weeks), no enzyme exhibited any decrease in activity after one year at ambient temperature, while all enzymes were stable at 4°C. Medium-Term Storage A wider variety of enzymes was also tested after storage at -20°C and ambient temperature for one week (Figure 2). All tested enzymes exhibited similar levels of digestion after storage at the two different temperatures. Exceptions to this were AluI and BglI, which apparently failed to digest after storage at -20°C. Repetition of these digests confirmed that this was an experimental error because both repeated digests (-20°C and ambient) appeared identical (results not shown). Ambient Transport Conditions To test the stability of these enzymes under genuine travel conditions, a selection was sent by conventional airmail to both America and Africa. After the return journey to America, activity was examined using end-point titration as a sensitive assay of enzymatic activity (Figure 3). EcoRI was slightly affected

Figure 2. Ethidium bromide-stained 1% agarose gel comparing the stability of a selection of restriction enzymes after storage for one week at room temperature or -20°C. Enzymes are arranged alphabetically from left to right, as indicated in the legend. λ DNA (250 ng) was digested for 1 h with 1 µL enzyme. The specific activity of the enzymes varied slightly, but all were in the region of 10 U/µL. 540 BioTechniques

by the journey, although the enzyme had to be diluted greater than tenfold before this loss of activity became apparent compared to the -20°C control. No discernible loss in activity for HindIII could be seen, with complete λ digestion even at a dilution of 1/100. There was a marginal loss in the activity of SmaI, but this could only be seen at a dilution of 1/100, a surprising finding given the thermolabile nature of this enzyme. HindIII and PstI were sent by conventional airmail to Africa, and activity was tested in situ following arrival. Activity was noted following a 1-h incubation, although better digestion was observed after overnight incubation (Figure 4), suggesting some loss of activity had occurred during transport. HindIII transported to the laboratory via a conventional cold chain (i.e., overland transport on ice from South Africa) was used as a positive control. There was no discernible difference in the ability of the two samples of enzyme to digest λ DNA after overnight incubation, suggesting that transport by conventional mail may be a viable alternative to refrigeration as a means of delivering restriction enzymes to laboratories in isolated regions. As the enzymes tested here represent only a small fraction of those commercially available, this work can only be considered preliminary. However,

Figure 3. Ethidium bromide-stained 1% agarose gels showing the effect of transport on enzyme stability. The lower lanes show the results of digestion with an enzyme that had been posted to Boston, MA, USA and returned to our laboratory (Edinburgh, UK) by conventional airmail (transit time six days). The upper lanes show digestion with enzymes that had been stored at -20°C for the same time. In both cases, 250 ng λ DNA was digested with 1 µL enzyme at 10 U/µL for 1 h. Serial dilutions of enzymes (up to 1/100) were used so that relative enzyme activity could be determined. Undigested λ DNA was used as a positive control. Vol. 29, No. 3 (2000)

Short Technical Reports all enzymes tested by us under the various conditions have shown a surprising degree of stability when stored as suggested by the manufacturers, without any further modifications or additions. These data suggest that standard restriction enzyme storage buffers are remarkably effective at maintaining activity, most likely because of the 50% glycerol in the buffer. Our results suggest that EcoRI is stable for at least one year as supplied at room temperature. Uritani et al. (4) reported that EcoRI was only stable for one week at room temperature following desiccation with trehalose, which suggests that this treatment may actually decrease activity (although EcoRI that was desiccated without trehalose exhibited even less activity, leading them to conclude that trehalose treatment was beneficial). Our results would suggest that storage without desiccation is even better. Contributory factors towards this apparent stability may be that these enzymes are usually provided at concen-

trations much higher than is necessary for standard digestions, as is indicated by the ability of HindIII to digest λ DNA at dilutions of 1/100 (Figure 3). It is likely that a percentage of enzyme activity was lost during transit to Africa, but enough remained to allow digestion. Alternative approaches may be to recalibrate activity after arrival, add more enzyme or simply to extend the incubation period (Figure 4). Although we have only tested restriction enzymes, these findings may well be applicable to other classes of enzymes, particularly thermostable polymerases. Cold-chain transport to countries such as Tanzania is excessively expensive at present—$800–$1000 US for airmail directly from Europe or $600 US from South Africa. Enzymes posted by airmail cost less than $1 US from the UK.

The research needed to unequivocally confirm these findings would not be practical to perform in our laboratory, but a more detailed investigation of a greater range of restriction enzymes by the manufacturers themselves may benefit those in the research community in countries where enzymes are currently difficult or impossible to obtain because of transportation or storage constraints. REFERENCES 1.Colaço, C., S. Sen, M. Thangavelu, S. Pinder and B. Roser. 1992. Extraordinary stability of enzymes dried in trehalose: simplified molecular biology. Biotechnology (NY) 10:1007-1011. 2.Lee, Y.H., R.W. Blakesley, L.A. Smith and J.G. Chirikjian. 1978. Preparation and properties of insolubilized restriction endonucleases. Nucleic Acids Res. 5:679-689. 3.Rossi, S., M.P. Buera, S. Moreno and J. Chirife. 1997. Stabilization of the restriction enzyme EcoRI dried with trehalose and other selected glass-forming solutes. Biotechnol. Prog. 13:609-616. 4.Uritani, M., M. Takai and K. Yoshinaga. 1995. Protective effect of disaccharides on restriction endonucleases during drying under vacuum. J. Biochem. (Tokyo) 117:774-779.

This work was supported by a grant from the Animal Health Program of the UK Department for International Development. Thanks are due to Lughano J.M. Kusiluka for his help in facilitating the Tanzanian experiments. Address correspondence to John B. March, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK. e-mail: [email protected] Received 21 July 1999; accepted 10 May 2000.

Figure 4. Ethidium bromide-stained 0.8% agarose gel of λ DNA (3.2 µg) digested with restriction enzymes after transit under different conditions. HindIII and PstI (lanes 2 and 3) were transported to Africa by conventional airmail (transit time 21 days). Control HindIII was transported to the same laboratory using a conventional cold chain. All digests were performed overnight at 37°C in a volume of 20 µL with 2 µL enzyme and 2 µL 10× buffer as recommended by the manufacturer. Lane 1 contains a 1-kb ladder, and lane 4 shows undigested λ DNA that was run as a positive control.

Jason Clark, Jennifer C. Harrison, Robinson H. Mdegela1 and John B. March Moredun Research Institute Penicuik, Scotland, UK 1Sokoine University of Public Health Morogoro, Tanzania

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