M. McDonald , Morag S. McDonald , Nik Norulaini Ab. Rahman , Graham Williamson. aBower Building .... experimenter may produce continuous banding pat-
Journal of Microbiological Methods 32 (1998) 213–215
Journal of Microbiological Methods
Examination of the pipetting variation between individuals performing random amplified polymorphic DNA (RAPD) analysis a, a a ,b a a ,c Neil R. McEwan *, Jehan Bakht , Edi Cecchini , Allan Coultart , Chiara Geri , Fiona a a a ,d M. McDonald , Morag S. McDonald , Nik Norulaini Ab. Rahman , Graham Williamson a a
Bower Building, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8 QQ , Scotland b Dipartimento di Biologia delle Plante Agrarie-Sazione di Genetica, Via Matteotti I /B, Pisa, Italy c I.M.D.C.N.R., Via Svezia 10, Pisa, Italy d School for off Campus, Universiti Sains Malaysia, USM, 11800, Penang, Malaysia Received 1 July 1997; received in revised form 15 August 1997; accepted 9 September 1997
Abstract The similarity between random amplified polymorphic DNA (RAPD) profiles generated by nine experimenters is presented. Despite simultaneously using the same pipettes, the same solutions and the same thermocycler, no two RAPD profiles generated identical banding patterns. This result suggests that before using a particular primer, it is essential that all experimenters should obtain a reference profile for their own work, rather than comparing it to one generated by another researcher. 1998 Elsevier Science B.V. Keywords: Random amplified polymorphic DNA (RAPD) profile; Simultaneous experiment; Thermocycle
1. Introduction Random amplified polymorphic DNA (RAPD) analysis is becoming one of the major methods used for strain identification of microorganisms. It is already well documented that there is considerable variation in the RAPD banding pattern between different laboratories, sources of the enzyme Taq polymerase, differences between PCR machines within a single laboratory (Penner et al., 1993; Schierwater and Ender, 1993) and differences in the PCR cycling time (Yu and Pauls, 1992). In addition, it is well known that variations in the concentration of components of the reaction cocktail can affect the products. Of particular importance appears to be the
variation in magnesium ion concentration, where bands on a RAPD profile can appear or disappear when the Mg 21 is changed from 1.5 mM to 4 mM (Benter et al., 1995), however variation in the concentration of other components of the reaction cocktail may also affect the results (Innis et al., 1988). To the knowledge of the authors, there has been no investigation of the banding patterns obtained by a number of scientists using the same thermocycler and the same reagents at the same time. This paper demonstrates that considerable variation may also result from different scientists performing RAPDs simultaneously. 2. Materials and methods
*Corresponding author. 0167-7012 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 97 )00086-9
Stock reagents were prepared to the correct con-
214
N.R. McEwan et al. / Journal of Microbiological Methods 32 (1998) 213 – 215
centration by one of the authors (McEwan) prior to each experimenter adding them to the reaction cocktail. All components of the reaction cocktail were kept on ice after being prepared, and after each author prepared a reaction mix. This too was returned to the ice bucket until all nine samples had been prepared. The PCR reagents were added in the following order, and each was mixed into the solution before addition of the next component; 76.5 ml of distilled water, 10 ml of 310 reaction buffer (500 mM KCl, 100 mM Tris.HCl (pH 8.3), 15 mM MgCl 2 ), 2 ml 10 mM dATP, 2 ml 10 mM dCTP, 2 ml 10 mM dGTP, 2 ml 10 mM dTTP, 4ml 50 mM oligonucleotide primer CCCACACCAC, 0.5 ml (5 Units / ml) Taq polymerase, 1 ml of intact E. coli cells (strain NM621) diluted to A 600 50.0006. An overlay of 50 ml of mineral oil was added. All reagents were added using either 10 ml (accuracy 0.5–10.0) or 100 ml (accuracy 20–100) pipettes, as applicable. The same pair of pipettes were used by all authors. After preparation of the PCR cocktail by individuals all further work was performed by a single author (McEwan). The cocktail was passed through 40 cycles of amplification; denaturation at 948C for 1
minute, annealing at 308C for 1 minute and extension at 728C for 2 minutes. A 10 ml aliquot of the reactant from each tube was removed and used for electrophoresis on a 4% (w /v) TBE (89 mM Tris.HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA) agarose gel. The gel was run at 7 V/ cm (i.e. 100 V when the distance between the two electrodes was 14 cm) and stained with 0.5 mg / ml (w /v) ethidium bromide (Maniatis et al., 1982).
3. Results and discussion The resulting gel is seen in Fig. 1. Clearly there are differences in the banding patterns obtained by the different scientists. This is reflected in both the number of bands observed, band intensity, and also in the molecular weights of these bands. Since the samples being investigated were all prepared for simultaneous amplification, there was no opportunity for variation due to differences in concentration of stock reagents, pipettes used, and reaction times in the cycler. The thermocycler and RAPD primer had been routinely used by one of the
Fig. 1. An electrophoretogram of the products of the RAPD reaction after electrophoresis on a 4% [w /v] TBE gel. The lanes are reactions prepared as follows (a) Edi Cecchini, (b) Graham Williamson, (c) Nik Rahman, (d) Morag McDonald, (e) Jehan Bakht, (f) Fiona MacDonald, (g) Chiara Geri, (h) Neil McEwan, (i) Allan Coultart and (m) 50 bp Molecular Weight Markers [Hoefer].
N.R. McEwan et al. / Journal of Microbiological Methods 32 (1998) 213 – 215
authors, without variation in banding patterns, for work described earlier (McEwan and Wheeler, 1995). In addition, other authors routinely used the thermocycler without reporting aberrations in banding patterns reported. It is postulated that the differences between the RAPD products are due to the differences in volumes being pipetted by the different experimenters. Exactly what is the major contributor to these difference is uncertain, although magnesium ion concentration in the final cocktail would seem to be the most logical candidate, as difference in Mg 21 concentration are well documented as sources of differences in RAPD profiles (Benter et al., 1995). However, the work of Benter and co-authors (Benter et al., 1995) describes a major difference in the pattern when concentrations are changed from 1.5 mM to 4 mM. This change would require addition in excess of a two fold increase in the volume of the 310 reaction buffer being added to achieve this increase in the number of magnesium ions present. In turn the other components of the 310 buffer would also be present at an increased concentration, and may also play a significant role. It is also unlikely that such a large variation in volume being added would be likely to happen between all nine experimenters. Thus, we postulate that the differences being observed are actually cumulative differences for a number of the components, in the small variation generated by the pipetting technique of the nine authors. Hence, this paper suggests that although a single experimenter may produce continuous banding pat-
215
terns for a RAPD protile, a degree of difference may be obtained when performed by different scientists. It is therefore suggested that, in all RAPD experimental work, the scientist should establish their own strain banding pattern for a given primer as a reference point, rather than using a previous reference pattern established by another scientist.
References Benter, T., Papadopoulos, S., Pape, M., Manns, M., Poliwoda, H., 1995. Optimization and reproducibility of random amplified polymorphic DNA in human. Anal. Biochem. 230, 92–100. Innis, M.A., Myambo, K.B., Gelfand, D.H., Brow, M.A.D., 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436–9441. Maniatis T., Fritsch E., Sambrook J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, pp. 150–163. McEwan, N.R., Wheeler, C.T., 1995. PCR on the gram-positive organism Frankia without prior DNA extraction. Trends Genet. 11, 168. Penner, G.A., Bush, A., Wise, R., Kim, W., Domier, L., Kasha, X, Laroche, A., Scholes, G., Molnar, S.J., Fedak, G., 1993. Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories. PCR Methods Appl. 2, 341–345. Schierwater, B., Ender, A., 1993. Different thermostable DNApolymerases may amplify different RAPD products. Nucleic Acids Res. 21, 4647–4648. Yu, K., Paul, K.P., 1992. Optimization of the PCR program for RAPD analysis. Nucleic Acids Res. 20, 2606.
