bacterial DNA isolation

1. ISOLATION OF DNA
FROM BACTERIAL CELL
K.ACHYUTH
2022-11-113
Msc plant pathology1

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
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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
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4. BACTERIAL DNA VS PLASMID DNA
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5. PRINCIPLE
The procedure of genomic DNA extraction can be divided into 4 stages:
1.A culture of bacterial cell is grown and harvested.
2.The cells are broken open to release their contents.
3.The cells extracted are treated to remove all components except the
DNA.
4.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.
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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
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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
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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
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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.
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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
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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
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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
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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)
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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
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15. DIFFERENT METHODS :
Bioling method
CTAB method
Stanadard phenol/chloroform
DNAzol
TE buffer
Proteinase k
silver nano particles
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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.
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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
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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
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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
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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
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21. References :
o 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.
o Chomczynski, P., Mackey, K., Drews, R., Wilfinger ,W., (1997). DNAzol: A
reagent for the rapid isolation of Genomic DNA. BioTechniques 22: 550-553
o 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
o 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
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22. Thank you
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