13.1. MOLECULAR BIOLOGY AND RECOMBINANT DNA METHODS
- Recombinant DNA technology allows the production of multiple copies of a single gene or DNA segment through gene cloning.
- The initial step involves extracting DNA mRNA/Plasmid from the cell and purifying it by separating it from other cellular components.
- Plasmids are circular, autonomously replicating DNA molecules, distinct from the bacterial genome and nonessential for cell survival under nonselective conditions.
- Removal of RNA from a DNA preparation is easily achieved by treating it with ribonuclease (RNase).
- Protein contamination can be eliminated by digesting it with a proteolytic enzyme like proteinase K.
- Centrifugation is commonly used for DNA purification, either for removing cellular debris or recovering precipitated nucleic acid.
- The supercoiled conformation remains stable only when both polynucleotide strands are intact, giving it the more technical name of covalently closed circular (ccc) DNA.
- If one polynucleotide strand is broken, the double helix reverts to its relaxed state, and the plasmid adopts an alternative conformation called open-circular (oc).
- Alkaline denaturation is a widely used method for separating plasmid from chromosomal DNA in bacterial cell extracts.
- The amount of UV radiation absorbed by a DNA solution is directly proportional to the DNA quantity in the sample.
- Absorbance is typically measured at 260 nm, where an absorbance of 1.0 corresponds to 50 micrograms of double-stranded DNA per ml.
- Electrophoresis is a technique that utilizes differences in electrical charge to separate molecules in a mixture.
- DNA molecules carry negative charges, causing them to migrate toward the positive pole in an electric field.
- Ethidium bromide staining is commonly used to visualize the results of a gel electrophoresis experiment.
- However, EtBr staining has limited sensitivity, and bands containing less than about 10 ng of DNA might not be visible after staining.
- By rephrasing and reorganizing the content, it has been made more unique and plagiarism-free while retaining the core information about molecular biology and recombinant DNA methods.
