June 09-41-2.indd - Kma.org.kw

More documents

Recommendations

Info

June 2009KUWAIT MEDICAL JOURNAL 117Original ArticleHeat Treatment of Bacteria: A Simple Method ofDNA Extraction for Molecular TechniquesAli A Dashti 1 , Mehrez M Jadaon 1 , Abdulsamad M Abdulsamad 2 , Hussein M Dashti 31Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Health Science Center, Kuwait University, Kuwait2Department of Surgery, Amiri Hospital, Kuwait3Department of Surgery, Faculty of Medicine, Health Science Center, Kuwait University, KuwaitABSTRACTKuwait Medical Journal 2009; 41 (2): 117-122Objective: To evaluate the efficacy of two simplemethods involving use of heat for extraction ofbacterial deoxyribonucleic acid (DNA) be used inmolecular techniques like polymerase chain reaction(PCR), restriction fragments length polymorphism(RFLP) and DNA sequencing and compare themwith DNA extraction using commercial kits.Design: DNA extraction by improved alternativemethods and commercial kit.Setting: Microbiology Research Laboratory, Facultyof Allied Health Sciences, Kuwait University,KuwaitMaterial: Forty isolates of Klebsiella pneumoniaeIntervention: DNA was extracted from isolatesby either boiling for 10 minutes or microwaveirradiation for 10 seconds. For comparison, DNAwas also extracted using a commercial kit. Allextracted DNA samples were analyzed by PCR,RFLP and / or DNA sequencing of TEM and SHVgenes of the bacteria.Main Outcome Measures: Successful extraction ofDNAResults: PCR, RFLP and DNA sequencing gave theexpected results in all the DNA samples extractedby all the three methods (boiling, microwaveirradiation and the commercial kit). The resultswere qualitatively equivalent in all methods.Conclusion: Heat may be used to extract DNA fromK. pneumoniae which can be utilized successfully inperforming PCR, RFL and DNA sequencing.KEY WORDS: bacteria DNA extraction, DNA sequencing, PCR, RFLPINTRODUCTIONThe science of molecular biology has become anintegral part of all medical research fields includingbacteriology. Techniques including polymerasechain reaction (PCR), restriction fragment lengthpolymorphism (RFLP), hybridization techniquesand DNA sequencing are being extensively usedin identification and classification of differentbacterial species and subspecies. In fact, manybacterial strains are now classified based solely onmolecular characteristics [1-2] . Molecular techniquesin bacteriology usually start with bacterial DNAextraction and purification. A large number ofDNA extraction methods (performed manuallyor by automation) have been and are still beingdeveloped, each of which has its own advantages anddisadvantages. Many of these methods are based onthe traditional phenol-chloroform extraction method,which needs a variable number of reagents andequipment [3-4] . Moreover, several trials have beenmade to simplify the procedure for bacterial DNAextraction and purification. These methods tried tobreak the cells and release the DNA using certain lysingagents containing different chemicals like lysosyme,proteinase K, TWEEN20, sodium hydroxide/sodium dodecyl sulfate, guanidine isothiocyanate,and Triton X-100 [5-14] . In addition to chemical agents,physical factors have also been attempted includingheating, cooling, freezing, microwave irradiation,beads beating, magnetic field capturing, binding toglass beads, the use of ultrasound waves and passingthrough heat-exchanger coils and nylon filters [5-18] .Address correspondence to:Prof. Hussein M Dashti, Department of Surgery, Faculty of Medicine, PO Box 24923, 13110, Safat, Kuwait. Tel: (965)5319475, Fax: 965-25333098, E-mail: aad@hsc.edu.kw
118Heat Treatment of Bacteria: A Simple Method of DNA Extraction for Molecular TechniquesJune 2009Many have also used combinations of chemical andphysical methods [5-19] . Still, most of these methodsare laborious, time consuming and costly. In thelast two decades, many commercial kits have beendeveloped to extract bacterial DNA using simplersteps and a shorter time frame. Although they madethe DNA extraction process quicker, such methodsare costly and require several steps and reagents,and sometimes special equipment, to obtain thetarget DNA [14,20] .In this study, the authors have tried two verysimple methods that may be used to extractbacterial DNA using heat only in a very simplemanner. Using heat for bacterial DNA extractionis not new. High temperature exposure is knownto cause damage to cell membranes and cellwalls [14,16,20-22] . Jose and Brahmadathan reportedthat heating at 94 °C for two minutes was enoughto denature cell walls [16] . Low temperatureswere also observed to destroy cell walls andmembranes. Freezing induces crystallization ofwater inside cells which leads to destruction ofcytoplasmic structures [12,16,20] . In fact, Tell et al usedcycles of freezing and thawing to obtain bacterialDNA [12] . In practice, heating bacterial materialfor DNA extraction purposes was performed byboiling in a water bath or on hot blocks, or usingmicrowave ovens [5-16] . Microwaves can cause manydifferent biological effects; these are mainly dueto the heating process (thermal effects) but thereare also athermal effects on cellular material,which were thought to be due to acceleration andcollision of ions with other molecules, partitioningof ions, or altering the polarity of molecules inalternating electric fields [22-24] . In this study, theuse of heat has been improved in two simplifiedways to extract DNA from bacteria. To assess thesuitability of the extracted DNA for performingmolecular biology techniques, the extractedbacterial DNA was processed by polymerasechain reaction (PCR), restriction fragment lengthpolymorphism (RFLP) and DNA sequencing. Forcomparison, a commercial DNA extraction kitwas also used. These two methods, as well as thecommercial kit, were tried on Klebsiella pneumoniaeisolates harboring extended spectrum ß-lactamase(ESBL). ESBLs are mainly derived from TEM,SHV or CTX-M β-lactamases that have mutated toexpand their spectrum of activity to include thirdgeneration cephalosporins [1,25] . Although theywere first reported in Klebsiella species [26] , ESBLsare now also commonly found in Escherichia coliand they have also been found in other species ofEnterobacteriaceae [1] . To date, more than 130 TEMand more than 104 SHV derivatives have beenfound [1,2,25] .MATERIAL AND METHODSThe study was conducted in the microbiologyresearch laboratary of the faculty of Allied HealthSciences, Kuwait University. Approval of the localethical committee was obtained.SamplesForty strains of Klebsiella pneumoniae wereincluded in this study. These strains were isolatedfrom a variety of clinical specimens submitted tothe clinical bacteriology laboratories in Al-AmiriHospital. They were flagged as ESBL-positive bythe Vitek 2 GNI and AST-N020 cards (Bio Merieux,Marcy L’Etoile, France). Samples were grown at 37 °Con Luria Bertani (LB) media (from GIBCO, BRI, LifeTechnologies, UK), before extracting their DNA.Methods for DNA extractionIn the first method, two colonies of overnightgrowth bacteria were used. The colonies were putin a test tube containing one ml of distilled waterand boiled for 10 minutes in a water bath, and thenwere centrifuged for five minutes at 1000 rpm. Fivemicroliters of the supernatant were used for the PCR.The second method was based on using a Nationalmicrowave oven (Matsushita Electric IndustrialCompany, Japan) to heat the bacterial colonies (twocolonies dissolved in 500 μl distilled water) for 10seconds, followed by centrifugation for two minutesat 1000 rpm. Similarly, 5 μl of the supernatant wereused for the PCR.Commercial Kits for DNA extractionGenomic DNA from the same bacterial isolateswas extracted for PCR by using Gentra PuregeneDNA isolation kit (QIAGEN Inc., Valencia, CA,USA) according to the manufacturer’s instructions.DNA samples were tested by spectophotometery atdual UV light (260/280) and the ratio was 1.7-1.9 forall samples.PCRPCR was performed on all the DNA samplesextracted using the two methods and the commercialkit. Five microliters of the DNA were mixed with 45 μlof pre-aliquoted Reddy-Load PCR Mix (from ABgene,UK) containing 1.25 units of Taq DNA polymerase,75 mM Tris-HCl (pH 8.8), 20 mM (NH 4) 2SO 4, 1.5mM MgCl 2, 0.01% (v/v) Tween 20, 0.2 mM of eachof the four deoxynucleotide triphosphates (dATP,dCTP, dGTP and dTTP) and 100 pmol of each of theprimers indicated in Table 1. The expected sizes ofPCR products for the two sets of primers were 308and 858 base-pairs (bp), respectively (Table 1). ForSHV primers, the PCR mixture was incubated for fiveminute at 95 °C as an initial denaturation step, followed
Page 2 and 3: Vol. 41 No. 1KUWAIT MEDICAL JOURNAL
Page 4: KUWAIT MEDICAL JOURNALKUWAIT MEDICA
Page 8 and 9: June 2009KUWAIT MEDICAL JOURNAL 91E
Page 10 and 11: June 2009KUWAIT MEDICAL JOURNAL 93R
Page 12 and 13: June 2009KUWAIT MEDICAL JOURNAL 95O
Page 14 and 15: June 2009KUWAIT MEDICAL JOURNAL 97c
Page 16: June 2009KUWAIT MEDICAL JOURNAL 99p
Page 20 and 21: June 2009KUWAIT MEDICAL JOURNAL 103
Page 74 and 75: June 2009400 mg/kg/day for five day
Page 84 and 85:
June 2009KUWAIT MEDICAL JOURNAL 167
Page 86 and 87:
March 2009KUWAIT MEDICAL JOURNAL 16
Page 88 and 89:
Page 90 and 91:
Page 92 and 93:
Page 94 and 95:
Page 96 and 97:
Page 98 and 99:
Page 100 and 101:
show all

June 09-41-2.indd - Kma.org.kw

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?