DNA extraction from the most diffiult microorganisms including Mycobacterium, Staphylococcus, and Malassezia in fie minutes using sand
- DNA extraction from the most diffiult microorganisms including Mycobacterium, Staphylococcus, and Ma...
Staphylococcus, Mycobacterium, and yeasts have complex cell wallsthat impede cell lysis and the recovery of DNA using conventional extraction methods.Previously, we showed that sand particles effectively disrupt the cell walls of Staphylococcus and Mycobacterium for conducting DNA and RNA extraction. In this study,we aimed to test whether sand treatment of Staphylococcus, Mycobacterium, and Malassezia enables the extraction of usable DNA for polymerase chain reaction directly,without proteinase K, phenol-chloroform, and ethanol precipitation treatments. In ourprotocol, one or two colonies of each microorganism were mixed with sand particles in100 ml ddH 2O and vortexed for 3 minutes and the supernatant was used in the polymerase chain reaction protocol. The results showed that sand treatment of Staphylococcus, Mycobacterium and Malassezia allowed suffiient DNA to be extracted while theobtained DNA was pure enough to conduct polymerase chain reaction and restrictionenzyme digestion. We conclude that using sand for DNA extraction has important costadvantages while enabling DNA extraction to be completed in only fie minutes. Themethod’s simplicity also reduces the risk of contamination in studies involving manyexamples.
Obtaining sufficient quantities of pure DNA is crucial for polymerase chain reaction (PCR). DifferentDNA extraction protocols, such as phenol-chloroform, proteinase K, glass beads, thermal shockand boiling, have been used successfully for DNAisolation from gram-negative bacteria (1,2). However, Staphylococcus, Mycobacterium, and yeastshave complex cell walls that impede cell lysis and the recovery of DNA using conventional extractionmethods so the complex cell wall structure must bebroken down to isolate DNA or RNA from these microorganisms (3-6). For the Staphylococcus genus,such as gram-positive bacteria, this can be achievedby forming spheroplasts using lysostaphin, and forthe Mycobacterium genus by using chemicals likecetyltrimethyl ammonium bromide (CTAB) (4,7). In a previous study, we used sand particles to mechanically remove the cell wall of Staphylococcusand Mycobacterium for DNA or RNA extraction(8). In that study, after being vortexed for 3 minutes, the bacteria-sand mixture was treated usingthe proteinase K and phenol-chloroform, and ethanol precipitation protocols to obtain DNA while theguanidinium thiocyanate-phenol-chloroform protocol was followed for RNA extraction. Our sandmethod enabled sufficient amounts of pure DNAand RNA, which are usually difficult to obtain, tobe extracted from Staphylococcus, Mycobacterium.In addition, when we compared we found that farmore DNA was obtained using sand than glassbeads and the same amount of DNA was obtainedas the lysostaphin-treated microorganism.In this study, we investigated whether the proteaseK, phenol-chloroform, and ethanol precipitationprotocols are needed to obtain sufficient amountsof pure DNA for PCR using sand treatment alone.To test this, Staphylococcus, Mycobacterium andMalassezia were vortexed with sand particles for3 minutes before the supernatant was checked forDNA quantity and usability for PCR. The quality of the DNA template was also tested using the2kbp PCR protocol.
Sand was obtained from a natural stream (chosenbecause the edge structure of stream sand particles is sharper than that of sea sand) and sievedfor 0.5-3 mm size (Figure 1). The resulting finegrained sand was thoroughly cleaned with ddH2O to eliminate all dirt and dust without losing smallparticles autoclaving to sterilize it (9).Microorganisms and Growth ConditionsPreviously described methicillin-resistant Staphylococcus aureus (MRSA) containing the exfoliativetoxin A gene-encoding phage (9), Escherichia coliDH5α (E. coli) and, Mycobacterium tuberculosis H37Rv ATCC 25618 (10), Malesseiza furfur (CBS7,019) (11), strains were used in the study. LB(Luria-Bertani) Agar (Difco, USA) was used for toculture of the Staphylococcus and E. coli. Lowenstein-Jensen (LJ) (Becton, Dickinson, USA) agarmedia was used for the growth of the M. tuberculosis (10). The p63 (TP63), transcript variant 1 (ACCESSION NM_003722) cloned pcDNA-3 vector (previously constructed in our laboratory) wastransformed into the E. coli DH5α and grown on theLB agar containing ampicillin. Malassezia was inoculated in modified Leeming–Notman agar (MLNA)(1% w/v peptone, 1% w/v glucose, 0.2% w/v yeastextract, 0.8% desiccated ox bile, 0.1% v/v glycerol,0.05% w/v glycerol monostearate, 0.5% v/v Tween60, 2% v/v oleic acid, and 1% w/v agar in distilledwater) supplemented with cycloheximide (0.5%)and chloramphenicol (0.05 %). The culture was incubated at 32°C for 2 weeks (11).
DNA Extraction Using Sand
One or two colonies of each microorganism weremixed in 100 μl ddH2O with 100 mg sand. The bacteria-sand mixture was then vortexed at maximumspeed for 3 minutes before another 100 μl ddH2Owas added to the tube, mixed and centrifuged for 20 seconds at maximum speed. The supernatantwas collected for PCR. Ten μl of DNA from eachsupernatant sample was run and analyzed byethidium bromide (EtBr) (AppliChem, Germany)treated agarose gel electrophoresis.
Primers and PCR Protocol
The primers used in the study are presented in thetable.
Amplification was performed on a total of 50 μl containing 2 μl of the DNA template, 20 pmol of eachprimer, 2.5 mmol/L of the four deoxynucleotidesand 2.5 U of Taq polymerase (Thermo Scintific,Lithuania). The reaction mixtures were subjectedto 38 cycles of amplification in an S1000 Thermalcycler (BioRad, CA USA). The PCR machine wasprogrammed as follows: 94°C for 2 minutes to denature the template; 94°C for 45 seconds for denaturation; 60°C for 45 seconds for annealing; and68°C for 45 or 90 seconds for extension (dependingon PCR product length). After amplification, thePCR products were analyzed by EtBr treated agarose gel electrophoresis.
Restriction Enzyme Digestion
The DNA obtained from the microorganisms usingthe sand protocol were cut with the rare cutter enzyme HindIII (NEB, UK) and the frequent cutterenzyme HinfI (NEB, UK), according to the supplier’s recommended protocol before analysis by EtBrtreated agarose gel electrophoresis.
Two colonies from the MRSA and M. tuberculosis H37Rv ATCC 25618, E. coli DH5α and one colony from Malassezia were selected from the medium. After vortexing the individual sample with sand in 100 ml sterile ddH2O, another 100 ml H2O was added and briefly centrifuged. The supernatant was collected, and 10-15 ml of the supernatant was run on agarose gel. Figure 2 shows the individual DNAs run in the agarose gel. To investigate the usefulness of the obtained DNA, PCR was performed using individual supernatants obtained from the sand extraction protocol with specific primers for genes identified in the genome of the chosen MRSA and M. tuberculosis, Malassezia furfur from our previous studies (see Table 1). The figures 3, 4, and 5 show the PCR products from specific microorganisms. To test DNA integrity, the E. coli colonies transformed with pcDNA3-p63cDNA were treated with sand to obtain the supernatant. The PCR was performed using the two primer set available in our laboratory. One primer set covered the whole p63 cDNA, which is 2042 bp, while the second set was chosen from the forward and reverse side of the cloning site of the pcDNA-3-p63 vector plasmid, which produced 2321 bp PCR product. Figure 6 shows the results for the PCR products obtained from the first primer set covering the whole p63 cDNA and the results for the PCR products obtained from the second primer set which covers the p63 and parts of the pcDNA vector in addition to the whole p63 cDNA. The quality of the extracted DNA was also tested by restriction enzyme digestion. Figure 7 shows that the DNA obtained from the sand protocol was successfully digested by both the rare cutter enzyme HindIII and the frequent cutter enzyme HinfI
Because of their thick peptidoglycan layer, Gram-positive bacteria are resistant to the lysis required for DNA extraction while isolation of DNA from Mycobacterium, which also incorporate many complex lipids in their cell walls, is also difficult. Without disruption of the cell, standard extraction methods are ineffective so for these bacteria it is necessary to eliminate the cell wall for the cell to form a spheroplast. To transform Staphylococcus into its spheroplast form, lysostaphin is used while for Mycobacterium, CTAB is used to remove the cell wall. Similar to Staphylococcus and Mycobacterium simple lysis procedures, such as the use of sequential freeze-thaw cycles or incubation with hot detergent and proteases, have not produced high yields of DNA from many fungal species (12). In this report, we described an extremely easy method for DNA extraction from the most difficult microorganisms. In our previous study, we showed that a sand protocol effectively disrupts cell walls of Staphylococcus and Mycobacterium without the need for any chemicals. However, in the previous study, after the sand treatment, we treated the bacteria with proteinase K and phenol-chloroform, and ethanol precipitation protocols, which require considerable time to complete DNA extraction. Therefore, the present study investigated, firstly, whether cell wall disruption alone is enough to release DNA into the supernatant without proteinase K treatment, and secondly whether there is a PCR inhibitor effect of the sand treatment. Thirdly, we tested whether DNA integrity is affected by the sand treatment. Our results showed that using sand for DNA extraction enabled sufficient amounts of DNA to be extracted. The obtained DNA was also sufficiently pure to successfully conduct both PCR and restriction enzyme digestion. Finally, PCR products of over 2000bp length were obtained using Taq polymerase enzyme. The sand method uses the same physical mechanism as glass beads to disrupt bacteria cell walls mechanically. Because sand is a naturally occurring granular material composed of finely graded particles, we predicted that sand would be more effective than glass since the former’s surface is sharper and stronger than that of glass beads. Indeed in the previous experiment, we found that far more DNA was obtained using sand than glass beads (9). Some studies have described using silica and zirconium particles in addition to glass beads to disrupt bacteria cell walls as both particles, silica particularly, bind the DNA with high affinity (13). Although we didn’t investigate this in the present study, we would predict that using these materials together would yield less DNA than using the sand treatment. In addition, silica and zirconium need to be pre-treated with other chemicals before applying to DNA extraction. The sand method described in this study enabled sufficient DNA to be extracted from bacteria with rigid cell walls that normally make this process difficult. The obtained DNA is also sufficiently pure to successfully conduct PCR and restriction enzyme digestion. We, therefore conclude that using the sand method has important advantages, particularly decreasing costs and reducing the time need to extract DNA. Pre-prepared sand can be either used or stored almost indefinitely in the laboratory while our new method’s simplicity reduces the risk of contamination in epidemiological studies involving multiple samples. Acknowledgments: This work was supported by the resources of Medical Microbiology Department of the Ankara University Medical School Conflict of interest: None
1. Liu YT (2008) A technological update of molecular diagnostics for infectious diseases Infectious disorders drug targets8:183-188
2. Tan SC, Yiap BC (2009) DNA, RNA, and protein extraction: thepast and the present Journal of biomedicine & biotechnology2009:574398 doi:10.1155/2009/574398
3. Aldous WK, Pounder JI, Cloud JL, Woods GL (2005) Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitativereal-time PCR Journal of clinical microbiology 43:2471-2473doi:10.1128/JCM.43.5.2471-2473.2005
4. Amaro A, Duarte E, Amado A, Ferronha H, Botelho A (2008)Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacteriumavium subsp. avium Letters in applied microbiology 47:8-11doi:10.1111/j.1472-765X.2008.02372.x
5. Oliveira CF, Paim TG, Reiter KC, Rieger A, D’Azevedo PA (2014)Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates Revista do Instituto de Medicina Tropical de Sao Paulo 56:29-33 doi:10.1590/S0036-46652014000100004
6. Vingataramin L, Frost EH (2015) A single protocol for extractionof gDNA from bacteria and yeast BioTechniques 58:120-125doi:10.2144/000114263
7. van Belkum A, Bax R, Peerbooms P, Goessens WH, van Leeuwen N, Quint WG (1993) Comparison of phage typing and DNAfigerprinting by polymerase chain reaction for discriminationof methicillin-resistant Staphylococcus aureus strains Journalof clinical microbiology 31:798-803
8. Sahin F, Kiyan M, Karasartova D, Calgin MK, Akhter S, TuregunAtasoy B (2016) [A new method for the disruption of cell walls ofgram-positive bacteria and mycobacteria on the point of nucleicacid extraction: sand method] Mikrobiyoloji bulteni 50:34-43