INTRODUCTIONRAPD-PCR is means of creating a biochemical fingerprint of an organism. In standard PCR procedure a primer sequence is used specifically to amplify a known sequence of an organisms genome. In RAPD-PCR, however, random primer sequences may be used in organisms where a specific genome sequence is not known. Random parts of the organisms genome are produced, which are expected to be identical among related species, and thus similar banding patterns should be produced in gel electrophoresis.
This technology is proving to be quite useful in typing strains of bacteria involved in nosocomial (hospital acquired) outbreaks of infectious disease. Typing is particularly useful for new strains arising due to the use of broad-spectrum antibiotics from which certain organisms develop a resistance. Patients are generally more susceptible than the general public to these types of epidemics due to things such as pre or post surgical procedures, skin trauma (burns, wounds), catheterization, HIV, and prolonged stay.
The two RAPD primers used in this experiment were OPA-02 and OPA-06 for eight known control bacteria, which have previously produced good banding results, and for approximately 20 unknown bacteria; all bacteria identified under the result`s section.
MATERIALS AND METHODSIsolation of total cellular DNA was done using the Qiagen, Inc. protocol. Isolation of gram positive and negative bacteria was as follows:
Gram negative bacteria--1. Added 180ul buffer ATL to a 1.5ml tube.2. Using sterile technique transferred a loopful of bacteria to the 1.5ml tube and placed in the 37 C incubator for 30 minutes.3. Added 25ul of Proteinase K and placed in 70 C water bath for 30 minutes.4. Transferred to 95 C water bath for 30 minutes.
Gram negative bacteria--
1. Added 180 ul buffer ATL to labeled 1.5 ml tube.2. Using sterile technique transferred a loopful of bacteria to the 1.5 ml tube.3. Added 20 ul of Proteinase K and placed in a 55 C incubator overnight.
The isolation protocol, for both gram positive and negative bacteria, continued as follows:
1. Added 200ul buffer AL to the sample, mixed with vortexing, and incubated at 70 C for 10 minutes.2. Added 210ul of ethanol (EtOH) to each sample and mixed well.3. Poured into a spin column that was placed on a collecting tube. Labeled the spin column.4. Centrifuged at 10K rpm for 1 minute.5. Discarded the collecting tube and replaced it with a new collecting tube.6. Added 500ul buffer AW to each spin column, which was then spun at 10K rpm for 1 minute.7. Removed the contents of the collecting tubes.8. Added another 500ul of buffer AW to each spin column.9. Centrifuged at full speed for 3 minutes.10. Discarded the collecting tube and its contents and replaced it with a new collecting tube.11. Added 200ul buffer AE to each spin column and incubated for 1 minutes at room temperature. 12. Centrifuged for 1 minutes at full speed.13. Removed the 200 ul from the collecting tube and loaded it back onto the spin column where it was centrifuged again at full speed for 1 minutes.14. Transferred the 200ul solution of isolated nucleic acids to a clean 1.5 ml tube and labeled it.
RAPD PCR Analysis--
1. Dispensed 23ul of RAPD primer solution into each PCR reaction tube.2. Pipetted 2ul of each isolated DNA template into reaction tubes.3. Mixed the contents of the tubes until the beads disappeared.4. Pulse spun all tubes in the microcentrifuge 5. Reactions were then placed into the thermal cycler and run according the following profile:
1 cycle at 94 C for 4 minutes (initial denaturation). 45 cycles at 94 C for 30 sec (denaturation), 36 C for 30 sec (annealing), and 74 C for 1 minute (extension) 1 cycle at 74 C for 4 minutes (final extension) 1 cycle at 4 C and held
6. Prepared four agarose 20 lane MIDI gels.7. Added 6ul 5X- sample buffer to each reaction tube.8. Loaded 15ul of each sample into the gels9. Loaded in each of the four gels were 12ul DNA 123 marker and control samples.
RESULTSResults of gel electrophoresis are shown (see Gel #1b, #2b, #3b, #4). OPA-06 was not shown to be as effective of a primer as OPA-02. Using OPA-06, unknown bacteria 4, 11, 14, 22, 19, 46, 16 and control samples 53, 116, 132, and 146 failed to produce any discernable bands. Data from OPA-06 could not be used to produce a phylogenetic tree. All but three samples produced good banding results using OPA-02 primer. Identities of the unknown samples are as follows:
1 Bacillus subtilis2 Pseudomonas aeruginosa3 Staphylococcus4 Bacillus megaterium5 Mycobacterium6 Proteus vulgaris7 Serratia marcescens8 Bacillus cereus9 Neiserria10 E. Coli11 Enterococcus12 Bacillus subtilis 13 P. aerugenosa14 Staphylococcus16 Mycobacterium17 P. vulgaris18 Serratia marcescens19 B. cereus22 Enterococcus Controls: 132- Enterobacter; 146- Acinotobacter; 107- Enterococcus; 26- Klebsiella; 53- P. aeruginosa; 62- P. vulgaris; 117- Staphylococcus aureus; 116- Staphylococcus
The binary system used is shown in table 2 (1 = band is present, 0 = band not present). Figures 1-4 are the phylogenetic trees produced from the data contained in tables 1 and 2. The numbers shown between each fork represent confidence values for the relationship of the two species. The higher the value, the greater the confidence.
DISCUSSIONAlthough electrophoresis produced good visible results, the phylogenetic results were not what would be expected. DNA from morphologically similar species would be expected to be in the same branching. For instance, unknown 18, a bacillus but present on the same fork as unknown 117, a cocci. Unknown 1 and 8 for example, are both rod shaped bacteria but are on opposite side of the tree. Unknown 12 and 13 are found on the same fork, but both have opposite gram reactions, and thus different cell wall structures; a close relationship would not be expected. Some organisms are present more than once such as Staphylococcus (unknown 3 and 14) but are not anywhere near one another on the tree. Even with intraspecies differences they would still be expected to occupy the same branch. Contamination from such things as dead skin or other airborne organisms during the isolation process is likely to produce invalid results.
Only OPA-02 produced useable fragments, which were used for phylogenetic analysis. To improve results, a greater number of different primers might be used. One experiment involving RAPD analysis of Acinetobacter strains (Koeleman et. al., 1998) recently showed that at least five different primers were needed out of the six they initially used to produce optimal RAPD fingerprinting. However, in another experiment involving the typing of Borrelia burgdorferi, four primers were used and were said to be reproducible up to the 95% similarity level (Wang et. al., 1998).
In the Acinetobacter study it was also noted that although RAPD technique is a simple, rapid method of typing, its lack of reproducibility has been reported. According to the article, this was due to the method`s susceptibility to variation by primer and DNA concentration, DNA template quality (addressed above concerning contamination during the DNA isolation procedure), gel electrophoresis, and type of DNA polymerase. Standardization concentrations of purified genomic DNA, and standardized RAPD kits, helped to show RAPD as a reliable typing method.
In another article differentiating Leuconostic mesenteroid strains (Holt et. al., 1998) according to the type of Dextran production was utilized. This experiment involved a modification of the RAPD-PCR method. The modification was to design RAPD primers, which would produce fragments within conserved sequences of certain genes to determine difference among identical species.
REFERENCESWang, G., van Dam, A. P., Spanjaard, L., Dankert, J. 1998. Molecular typing of Borrelia burgdorferi sensu lato by random amplified polymorphic DNA fingerprinting analysis. J. Clin. Microbiology. 36: 768-776
Koeleman, J.G.M., Stoof, J., Biesmans, D.J., Savelkoul, P.H.M., Vandenbroucke-Grauls, C.M.J.E. 1998. Comparison of amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism fingerprinting for identification of Acinetobacter genomic species and typing of Acinetobacter baumannii. J. Clin. Microbiology. 36: 2522-2529
Holt, S.M., Cote, G.L. 1998. Differentiation of dextran-producing Leuconostoc strains by a modified randomly amplified polymorphic DNA protocol. Applied Env. Microbiology. 64: 3096-3098