ISOLATION OF DNA FROM BACTERIAL CELL 1.pptx

bacterial DNA isolation

1. ISOLATION OF DNA FROM BACTERIAL CELL K.ACHYUTH 2022-11-113 Msc plant pathology 1

2. INTRODUCTION : The isolation and purification of DNA is a key step for most protocols in molecular biology studies and all recombinant DNA techniques (Sambrook et al ., 1989) The aim of the present study was to develop a simple, rapid and inexpensive protocol of ultra pure bacterial genomic DNA extraction suitable for use in molecular methodology DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods 2

3. Why isolate DNA from bacteria ? Understanding Bacterial Genetics The isolation of DNA from bacteria allows us to study their genetic makeup, including gene expression and genetic mutations, and how these processes relate to bacterial growth and resistance Biotechnology Isolated DNA from bacteria has been used in diverse applications, including the production of antibiotics, recombinant proteins, and genetically modified crops 3

4. BACTERIAL DNA VS PLASMID DNA 4

5. PRINCIPLE The procedure of genomic DNA extraction can be divided into 4 stages: A culture of bacterial cell is grown and harvested. The cells are broken open to release their contents. The cells extracted are treated to remove all components except the DNA. The resulting DNA is then controlled. The cell is lysed by adding guanidium thiocyanate and a detergent comprising solution A. It is then centrifuged to separate the RNA and proteins. The resulting supernatant mainly consists of genomic DNA and sometimes RNA. The DNA is precipitated using alcohol. 5

6. Materials required : Laboratory gloves, racks, pipettes, flasks, tubes spectrophotometer with UV-Vis measurement range, centrifuge, bacterial culture Chemicals : Nacl , SDS, proteinase k, sodium acetate, phenol - chloroform mixture, TE buffer, ethanol-70%,isopropanol etc 6

7. Procedure : A 48 h. old single colony of the bacteria was grown on medium and was inoculated into 100 ml of sterilized medium The inoculated broth was incubated overnight (12 h.) in an orbital shaker (150 rpm) at room temperature The broth (2 ml) was taken in micro centrifuge tube and centrifuged at 10,000 rpm for 10 min. at 4°C The supernatant was decanted and the pellet was washed twice with 1.5 ml of NaCl(1%) to remove extra cellular polysaccharides 7

8. The cells were then suspended in 875 µl of TE buffer into which 100 µl of SDS and 5 μl of proteinase K were added and incubated at 37°C for 1h. After incubation, equal volume of phenol-chloroform mixture was added and incubated for 5 min. The contents were then centrifuged at 10000 rpm for 10 min at 4°C. After centrifugation, three layers were observed. Top aqueous layer containing DNA, a middle layer with cell debris and a bottom layer containing phenol 8

9. The top aqueous layer was carefully pipetted out and transferred in to a fresh tube and the process was repeated once again. After centrifugation, the supernatant was collected and 100 µl of 5 M sodium acetate was added and mixed gently. Isopropanol (2 ml) was added and mixed gently by inversion till DNA precipitates as white thread-like strands. 9

10. The reaction mixture was then incubated at -20 °C for 2 h. After incubation, it was centrifuged at 10000 rpm at 4°C for 10 min, and the supernatant was discarded carefully. The DNA pellet was then washed with 70 per cent ethanol, followed by washing with 100 per cent ethanol. The DNA pellet was then air dried and dissolved in distilled water 10

11. Qualitative analysis of DNA using agarose gel electrophoresis : Agarose solution (0.8%, 100 ml I X TAE buffer ),The solution was allowed to cool and 2-3 drops of ethidium bromide was added and mixed well. Gel casting tray was wiped with 70% ethanol and warm agarose solution was then poured and allowed to solidify. After solidification, the casting tray was placed in the electrophoresis unit filled with I X TAE buffer in such a way that the gel is immersed in the buffer 11

12. Sample (5 µl) was mixed with loading dye (1 µl) and this mixture was loaded into the wells except the first well to which 1Kbp ladder was added Electrophoresis was carried out at 80 V till the dye has moved to two third of the gel and the bands of DNA were visualized using gel documentation system 12

13. Bacterial genomic DNA isolation using sonication for microarray analysis The sonication method for DNA isolation is based on the use of high frequency sound waves, which create shear forces that break open the outer cell membrane of bacteria. The intracellular genomic DNA is then extracted and purified, resulting in high-quality DNA suitable for microarray analysis (Zhang et al ., 2005) 13

14. Harvest Bacterial Cells Cell Lysis Sonication DNA Extraction Purification Precipitate DNA DNA Pellet Formation Wash DNA Pellet Dry and Resuspend DNA Assess DNA Quality and Quantity Microarray Analysis 14

15. DIFFERENT METHODS : Bioling method CTAB method Stanadard phenol/chloroform DNAzol TE buffer Proteinase k silver nano particles 15

16. DNA ZOL : Guanidine thioisocyanate present in DNAzol is capable of binding DNA to silica particle column. Subsequently the silica with adsorbed DNA is washed to remove impurities and the clean DNA eluted in appropriate buffer . The DNAzol procedure is based on the use of a novel guanidine-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from a cell lysate . 16

17. Material and methods : Strains used: gram+ve : Enterococuss , Staphylococcus gram – ve ; Listeria Staphylococcus lugdunensis was grown aerobically in trypticase soy broth (TSB) ( Difco ) at 37°C Procedure : Staphylococcal cell pellets were resuspended in 100 µl buffer containing lysostaphin (20 g/ml), mixtures were incubated at 30 0c for 30 min now added 500 µl dna zol to lysed mixture and incubated at 65 0c for 5 min centrifused at 10000 rpm for 60sec and columns were with 70% ethanol 17

18. To elute clear dna 50 µl of warm TE buffer expect remain for 1 min centrifugation for 1min at 10000 xg quantity and purity checked in spectrophotometry Conclusion : Technique also eliminates the need for time-consuming organic extractions and ethanol precipitation, Finally, the modified protocol increase laboratory efficiency 18

19. Chemically synthesized silver nanoparticles : Ag is a toxic metal and has antibacterial agent against bacteria Nanotechnology has witnessed Ag with nano particles against + ve and – ve bacteria Silver nano particles has pronounced effect on metabolic activity and cell membrane damage synthesised AgNps cell lysis phenol-chloroform extraction ethanol ppt to extract DNa 19

20. TE buffer : Lysis will depend on freezing (-20 to -30 0c) and dry heating 90 to 95 0c Precipitation done at 13000 rpm Pure dna checked using nano drop spectrometry 20

21. References : Ausbel ,F.M., Brent, R., Kingston, R .E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl ,K., (1995). Current Protocols in Molecular Biology. John Wiley and Sons, 2.4.1. Chomczynski , P., Mackey, K., Drews, R., Wilfinger ,W., (1997). DNAzol : A reagent for the rapid isolation of Genomic DNA. BioTechniques 22: 550-553 Zhang, L., Foxman, B., Gilsdorf , J.R. and Marrs, C.F., 2005. Bacterial genomic DNA isolation using sonication for microarray analysis.  Biotechniques ,  39 (5), p:640-644 Goswami, G., Boruah, H., Gautom , T., Hazarika, D.J., Barooah, M. and Boro, R.C., 2017. Chemically synthesized silver nanoparticles as cell lysis agent for bacterial genomic DNA isolation.  Advances in Natural Sciences: Nanoscience and Nanotechnology ,  8 (4), p.045015 21

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