5 public questions tagged with this topic.
Lysozyme breaks down the peptidoglycan cell wall of bacteria to release DNA for isolation.
Ref: NCERT Class 12 Biology Chapter 9: Biotechnology: Principles and Processes Processes - Isolation of DNA and Gel Electrophoresis
Ribonuclease (RNase) degrades RNA, allowing pure DNA to be extracted during DNA isolation.
Ref: NCERT Class 12 Biology Chapter 9: Biotechnology: Principles and Processes Processes - Isolation of DNA and Gel Electrophoresis
Crude cell lysate contains proteins, lipids, RNA, and DNA. Phenol extraction, often with chloroform, is the deproteinization step. Phenol, being organic and hydrophobic, denatures proteins by disrupting hydrophobic interactions, causing them to unfold, partition into organic phase, or accumulate at interphase. RNase treatment may be included but phenol itself removes most proteins and also extracts residual lipids. Chelation by EDTA, mechanical homogenization, and alcohol precipitation do not selectively remove proteins and RNA; alcohol precipitates nucleic acids together while phenol specifically purifies away proteins effectively.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
Ethanol precipitates DNA by altering solvent properties. Water has high dielectric constant of 80, shielding electrostatic attraction between negative phosphate groups and positive cations like Na+. Ethanol has dielectric constant around 24, dramatically lowering solvent polarity when mixed. This allows sodium ions from added salt to neutralize phosphate charges efficiently, reducing hydration shell and DNA solubility. DNA becomes less hydrophilic, aggregates, and precipitates. Ethanol does not lower pH significantly, increase hydrophilicity, or break disulfide bonds; its action is purely through dielectric and solubility reduction enabling aggregation.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
EDTA (ethylenediaminetetraacetic acid) is a chelating agent essential in DNA isolation buffers. It binds divalent cations like Mg2+ and Ca2+ with high affinity, forming stable coordination complexes. Mg2+ is a crucial cofactor for DNases that degrade DNA and for nucleases that destabilize membranes. By sequestering Mg2+, EDTA irreversibly inhibits endogenous nuclease activity, protects genomic DNA from enzymatic degradation, and helps destabilize cell walls by removing membrane-stabilizing cations. It does not denature proteins directly, bind nucleic acids specifically, or degrade RNA; its function is strictly chelation-mediated protection and enzymatic inhibition.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
