Cells act as protective capsules, housing DNA, RNA, proteins, lipids, and other biomolecules. To study or manipulate these molecules, it is necessary to break open the cells a process known as lysis. This step is followed by isolating and purifying the molecule of interest.
The process varies depending on the type of cells or tissues being handled:
- Bacteria: Require harsher treatments due to their rigid cell walls.
- Plant Cells: Need mechanical or enzymatic approaches to penetrate their cellulose-based walls.
- Animal Cells: Easier to lyse due to the lack of cell walls.
Steps in Breaking Up Cells and Tissues
1. Cell Harvesting
Before lysis, cells must be separated from the growth medium through centrifugation. Freshly harvested or frozen samples are preferred to prevent enzymatic degradation.
2. Lysis Techniques
The approach to lysis depends on the cell type:
Bacterial Cells:
- Use of chemicals like EDTA destabilizes the outer membrane by binding divalent cations.
- Lysozyme breaks down the peptidoglycan layer of the cell wall.
- Detergents like SDS solubilize membrane lipids, releasing intracellular components.
Plant and Fungal Cells:
- Require enzymatic digestion or mechanical grinding to penetrate their tough walls.
Animal Cells:
- Mild detergents suffice due to their simpler structure.
For complex tissues, homogenization is often used to break the tissue into smaller units before cell lysis.
3. Crude Extract Formation
After lysis, the resultant crude extract contains a mixture of DNA, RNA, proteins, and other biomolecules. This step often fragments chromosomal DNA, so gentler methods are required when intact DNA is needed.
Nucleic Acid Isolation
1. DNA and RNA Purification
RNA Removal from DNA Samples:
Ribonuclease (RNase) enzymes are used to degrade RNA without affecting DNA. Commercially available RNases ensure the absence of DNase contamination.DNA Removal from RNA Samples:
Deoxyribonuclease (DNase) is used to eliminate DNA traces from RNA preparations.
2. Protein Removal
Proteins in the cell extract are removed to prevent nucleic acid degradation and interference. Methods include:
- Phenol-Chloroform Extraction: Denatures proteins and separates them into the organic phase, leaving nucleic acids in the aqueous layer.
- Proteolytic Enzymes (e.g., Proteinase K): Breaks down proteins in a safer and simpler manner.
3. Nucleic Acid Precipitation
To concentrate the nucleic acid solution:
- Alcohols like ethanol or isopropanol are added, along with salts (e.g., Na+, K+), to form a precipitate.
- Centrifugation collects the precipitate, which can be washed with 70% ethanol to remove salts.
Key Considerations and Challenges
Minimizing Contamination:
- Use DNase-free RNase or RNase-free DNase to avoid cross-degradation.
- Work in a sterile environment to prevent external contamination.
Ensuring Integrity:
- Use fresh or frozen samples to reduce enzymatic activity that can degrade target molecules.
- Optimize lysis conditions to avoid excessive fragmentation of nucleic acids.
Handling Hazardous Chemicals:
- Phenol, a common reagent, is toxic and requires proper handling or safer alternatives like affinity chromatography.
Applications in Research and Biotechnology
Breaking up cells and tissues is essential for:
- Molecular Biology: Isolating DNA or RNA for cloning, sequencing, and PCR.
- Biotechnology: Producing recombinant proteins or studying gene expression.
- Drug Development: Investigating protein functions or designing therapeutic molecules.
Breaking up cells and tissues is a cornerstone technique in modern biology. Mastering these methods allows researchers to unlock the molecular secrets within cells, enabling breakthroughs in genetics, biotechnology, and medicine. By understanding the intricacies of cell lysis and nucleic acid isolation, scientists can continue to innovate and explore the complexities of life at the molecular level.