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June 2009KUWAIT MEDICAL JOURNAL 121cells, such as boiling or microwave irradiation, waswidely used to extract DNA molecules [5,7,9,10-13] .Still, many of the previous methods were eitherfollowed or preceded by enzymatic or detergenttreatment to obtain DNA for molecular techniques.Many companies have utilized the previousconcepts in producing commercial kits that couldbe used in extracting DNA from a variety of cellularmaterial [14,19] . Although providing simpler DNAextraction methods, such kits added extra costs toexperiments needing DNA extraction.In this study, simplified DNAextraction methods toproduce bacterial DNA samples were evaluated. Theaim was to minimize the time and the need for reagents,while still not affecting the quality of DNA extractedand the productivity of the subsequent moleculartechniques. The methods were based on using heatwithout adding any reagent. Heating bacterial material(suspended in distilled water without any otheradditions) was achieved by boiling for 10 minutes ormicrowave irradiation for 10 seconds. It was shownthat these two methods have provided enough DNAmolecules to perform subsequent molecular biologytechniques. The methods were tried on ESBL genes ofKlebsiella pneumoniae. The ESBL genes were detected byPCR amplification of the DNA sequences coding forblaTEM and blaSHV ESBL genes. PCR was successfulin all cases, giving the expected PCR amplicons. Thatwas additionally verified by performing the same PCRprotocol on DNA samples extracted from the samebacterial samples using a commercial DNA extractionkit. PCR amplicons were qualitatively equivalentin all experiments. Furthermore, and in the RFLPexperiment, digestion of TEM and SHV PCR productswith Sau3AI and NheI endonucleases, respectively,showed the same fragments and results in all the testedsamples whether the DNA used for PCR was extractedby the two methods introduced by the authors or usingthe commercial kit. Finally, DNA sequencing was alsosuccessful in all DNA extraction methods used in thisproject, giving the expected sequences.To compare with the work of this study, a limitednumber of researchers have also used boiling and/ or microwave irradiation to extract DNA withoutany reagents added. However, most of theseresearchers have boiled their samples or exposedthem to microwave irradiation for a time longerthan the presented method in this paper [14-15] orhave subjected their samples to multiple microwaveirradiation [6,15] . To the best of the authors’ knowledge,the report by Merk et al was the only one in whichthe samples were boiled for 10 minutes like in ourstudy [14] . Unlike this paper, their samples were bloodand lung tissue which were artificially infected withBurkholderia cepacia. In addition, their extracted DNAwas processed by PCR only. The present paper maybe the first to report using a 10-minute boiling, or asshort as 10-second microwave irradiation to extractbacterial DNA suitable to perform three essentialmolecular biology techniques; namely PCR, RFLPand DNA sequencing.CONCLUSIONIn conclusion, the presented methods (heattreatmentof bacteria) are very simple, cheap, quickand successful methods for DNA extraction frombacteria in order to be used directly in moleculartechniques, yielding excellent results as othermore complicated methods for DNA extractionand purification. The findings of this study mayprobably encourage trying the procedure on othertypes of biological specimens such as whole blood,culture cells, body fluids and hair.ACKNOWLEDGEMENTThe authors wish to thank the Shared FacilitiesLaboratory of the Health Sciences Center, KuwaitUniversity (project number GM 01/01), forperforming DNA sequencing for this project.REFERENCES1. Bradford PA. Extended-spectrum beta-lactamasesin the 21 st century: characterization, epidemiologyand detection of this important resistance threat. ClinMicrobiol Rev 2001; 14:933-951.2. Jacoby G, Bush K. Amino Acid Sequences for TEM, SHVand OXA Extended-spectrum and inhibitor resistant ß-lactamases. Lahey Clinic. (Accessed January 1, 2008 athttp://www.lahey.org/Studies/)3. Gross-Bellard M, Oudet P, Chambon P. Isolation ofhigh- molecular-weight DNA from mammalian cells.Eur J Biochem 1973; 36:32-38.4. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning:A Laboratory Manual. New York, Cold Spring HarborLaboratory Press, 1989.5. Porteous LA, Armstrong JL, Seidler RJ, Watrud LS. Aneffective method to extract DNA from environmentalsamples for polymerase chain reaction amplificationand DNA fingerprint analysis. Curr Microbiol 1994;29:301-307.6. Herman LM, De Block JH, Waes GM. A direct PCRdetection method for Clostridium tyrobutyricumspores in up to 100 milliliters of raw milk. Appl EnvironMicrobiol 1995; 61:4141-4146.7. Agersborg A, Dahl R, Martinez I. Sample preparationand DNA extraction procedures for polymerase chainreaction identification of Listeria monocytogenes inseafoods. Int J Food Microbiol 1997; 35:275-280.8. Marra MA, Kucaba TA, Hillier LW, Waterston RH.High-throughput plasmid DNA purification for 3 centsper sample. Nucleic Acids Res 1999; 27:e37.9. Skowronski EW, Armstrong N, Andersen G, Macht M,McCready PM. Magnetic, microplate-format plasmidisolation protocol for high-yield, sequencing-gradeDNA. Biotechniques 2000; 29:786-788,790,792.
122Heat Treatment of Bacteria: A Simple Method of DNA Extraction for Molecular TechniquesJune 200910. Agarwal A, Kumar C, Goel R . Rapid extraction of DNAfrom diverse soils by guanidine thiocyanate method.Indian J Exp Biol 2001; 39:906-910.11. Orsini M, Romano-Spica V. A microwave-based methodfor nucleic acid isolation from environmental samples.Lett Appl Microbiol 2001; 33:17-20.12. Tell LA, Foley J, Needham ML, Walker RL. Comparisonof four rapid DNA extraction techniques forconventional polymerase chain reaction testing of threeMycobacterium spp. that affect birds. Avian Dis 2003;47:1486-1490.13. Zhu K, Jin H, Ma Y, et al. A continuous thermal lysisprocedure for the large-scale preparation of plasmidDNA. J Biotechnol 2005; 118:257-264.14. Merk S, Meyer H, Greiser-Wilke I, Sprague LD,Neubauer H. Detection of Burkholderia cepacia DNAfrom artificially infected EDTA-blood and lung tissuecomparing different DNA isolation methods. J Vet MedB Infect Dis Vet Public Health 2006; 53:281-285.15. Lou YK, Qin H, Molodysky E, Morris BJ. Simplemicrowave and thermal cycler boiling methods forpreparation of cervicovaginal lavage cell samples priorto PCR for human papillomavirus detection. J VirolMethods 1993; 44:77-81.16. Jose JJ, Brahmadathan KN. Evaluation of simplifiedDNA extraction methods for emm typing of group Astreptococci. Indian J Med Microbiol 2006; 24:127-130.17. Elkin CJ, Richardson PM, Fourcade HM, et al.High-throughput plasmid purification for capillarysequencing. Genome Res 2001;11:1269-1274.18. Dederich DA, Okwuonu G, Garner T, et al. Glassbead purification of plasmid template DNA for highthroughput sequencing of mammalian genomes.Nucleic Acids Res 2002; 30:e32.19. Smith K, Diggle MA, Clarke SC. Comparison ofcommercial DNA extraction kits for extraction ofbacterial genomic DNA from whole-blood samples. JClin Microbiol 2003; 41:2440-2443.20. Strus M. Action of physical agents on microorganisms.Rocz Panstw Zakl Hig 1997; 48:263-268.21. Goodwin DC, Lee SB. Microwave miniprep of totalgenomic DNA from fungi, plants, protists andanimals for PCR. Biotechniques 1993; 15:438, 441-442, 444.22. Höfler G. Detection of bacterial DNA using thepolymerase chain reaction (PCR). Verh Dtsch GesPathol 1994; 78:104-110.23. Banik S, Bandyopadhyay S, Ganguly S. Bioeffects ofmicrowave - a brief review. Bioresour Technol 2003;87:155-159.24. Woo IS, Rhee IK, Park HD. Differential damage inbacterial cells by microwave radiation on the basisof cell wall structure. Appl Environ Microbiol 2000;66:2243-2247.25. Turner PJ. Extended-spectrum beta-lactamases. ClinInfect Dis 2005; 41:S273-S275.26. Kliebe C, Nies BA, Meyer JF, Tolxdorff-Neutzling RM,Wiedemann B. Evolution of plasmid-coded resistanceto broad-spectrum cephalosporins. Antimicrob AgentsChemother 1985; 28:302-307.27. Picard C, Ponsonnet C, Paget E, Nesme X, Simonet P.Detection and enumeration of bacteria in soil by directDNA extraction and polymerase chain reaction. ApplEnviron Microbiol 1992; 58:2717-2722.
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