Separation of genomic DNA from bacteria is a fundamental technique in molecular biology. The process helps obtain high-quality DNA.
The technique has a crucial role in various applications including cloning, sequencing, and genetic analysis.
For extracting high-quality DNA efficiently, there is a step-by-step approach. A systematic approach helps in minimizing contamination and degradation.
In this blog, we will understand the process of isolation of genomic DNA from bacteria. We will also look at the protocols and methods.
Explain The Isolation Of Genomic DNA From Bacteria Principle
The isolation of genomic DNA From the bacteria Principle involves a series of steps. The approach is a step-by-step procedure, that breaks open the bacterial cells to release the DNA. It purifies the DNA from other cellular components.
Newly separated genomic DNA should be of high purity and quality. Its high integrity ensures successful downstream applications like PCR, sequencing, cloning, and various molecular biology functions.
The systematic step-by-step approach ensures the better yield and quality of DNA. The process is a foundational technique applicable in genetic research and diagnostics.
Isolation Of Genomic DNA From Bacteria Procedure
The key steps involved in the isolation of genomic DNA are as follows:
- Cell Lysis – The cellular content released when the bacterial cell wall and membrane disrupts. The process of cell lysis involves using a combination of mechanical disruption (e.g., bead beating or sonication), enzymatic lysis (e.g., lysozyme to break down the cellular wall), and chemical lysis (e.g., SDS or other detergents to easily dissolve the membrane).
- Denaturation and Removal of Protein – To prevent the DNA from contaminating, the removal of proteins and other cellular debris happens in this process. The process involves the use of proteases. Proteases cause the breakdown of proteins. Adding phenol-chloroform to the solution can also help with the denaturation process. Protease and phenol-chloroform facilitate the separation of denaturation of proteins from the aqueous DNA solution.
- DNA Precipitation – The precipitation of the genomic DNA forms in the solution. The precipitate forms use alcohol (typically ethanol or isopropanol) in salt’s presence. In this step, the DNA precipitates and the soluble impurities separate.
- DNA Purification – This step involves the purification of the DNA by washing it with ethanol. The purpose of ethanol is to remove leftover contaminants. The washed DNA dissolves in a suitable buffer or water for further use.
Isolation Of Genomic DNA From Bacteria Protocol
Conducting the separation of genomic DNA involves using various materials and reagents. These components include:
- Bacterial culture (E. coli or other species)
- Resuspension buffer (e.g., TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
- Lysozyme
- Lysis buffer
- Proteinase K
- RNase A
- Phenol-chloroform-isoamyl alcohol (in a specific ratio)
- Isopropanol
- 70% ethanol
- Nuclease-free water or TE buffer
The process also includes various equipment like:
- Centrifuge
- Microcentrifuge tubes
- Vortex mixer
- Water bath or incubator
- Pipettes and tips
Here is the protocol for the isolation of genomic DNA:
- Culture Preparation – The bacterial culture grows overnight in a suitable medium (e.g., LB broth). If necessary, the process involves the use of appropriate antibiotics.
- Cells Harvesting – For harvesting cells, the overnight centrifugation of bacterial culture pellet at 12,000 x g for 2 minutes. Eliminate the supernatant and resuspend the pellet in a resuspension buffer (TE buffer).
- Cell Lysis – For the lysis process, the resuspended cells are added with lysozyme solution. Stir the mixture gently by pipetting. To continue to process of lysing the cells, incubate the mixture at 37°C for 30 minutes.
- Protein Denaturation – For denaturation, add 200 µL of lysis buffer to the cell lysate. Stir the mixture well by inverting the tube. Now, add 20 µL of proteinase K (20 mg/mL) along with 10 µL of RNase A (10 mg/mL) to the mixture. This mixture thus formed has to be incubated at 55°C for an hour.
- Phenol-Chloroform Extraction – Isolation of Genomic DNA from bacteria phenol/chloroform method lyses the cells. To lysate, use phenol-chloroform-isoamyl alcohol and vortex vigorously for about 30 seconds. The next step is to centrifuge at 12,000 x g for 5 minutes. Centrifugation should happen at room temperature. Now, transfer the upper aqueous layer carefully to a new microcentrifuge tube.
- DNA Precipitation – For precipitation, equal isopropanol must be added to the aqueous phase. The next step is to invert the tube to gently mix it. For the formation of a DNA precipitate, incubate the mixture at -20°C for 30 minutes. Centrifuge at 12,000 x g for 10 minutes at 4°C. Eliminate the supernatant.
- DNA Wash – Gently wash the DNA pellet with some ethanol. Wash it by gently inverting the tube several times. Now, centrifuge at 12,000 x g for 5 minutes at 4°C. Dispose of the ethanol.
- DNA Resuspension – For the removal of any remaining or leftover ethanol, Air-dry the DNA pellet for about 5-10 minutes. Mix the purified genomic DNA in 50-100 µL of nuclease-free water or TE buffer.
- Quality and Quantity Analysis – Use a spectrophotometer or a fluorometer to measure the concentration and purity of the separated DNA. Analyze the extracted DNA for checking integrity by running a small aliquot on an agarose gel.
The Link Between Genomic DNA Isolation and Genetic Testing
The process of extraction of genomic DNA has a crucial function in genetic testing. High-quality and High-integrity DNA is essential for accurate analysis in various genetic tests.
Genetic tests identify genetic changes, mutations, and markers associated with inherited diseases, traits, or conditions. Through the analysis of isolated genomic DNA, researchers and clinicians can detect specific genes.
The analysis helps with the diagnosis of genetic disorders, identifying carriers, predicting disease risk, and tailor personalized treatments. In this way, DNA extraction can have a direct impact on the reliability and success of genetic testing procedures.
Conclusion
Isolation of genomic DNA from bacteria protocol outlines the steps involved in isolating high-quality genomic DNA from bacterial cells.
The separated DNA is of high quality and is useful for various downstream applications. The applications include PCR, sequencing, and cloning. The step-by-step approach ensures high-purity DNA extraction essential for reliable genetic analysis.
