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#plasmid DNA

3 public questions tagged with this topic.

Prokaryotic Cell - Structure and Components

Two prokaryotic cells show the following features: Cell P is approximately 0.3 µm long and lacks a cell wall. Cell Q con

Mycoplasmas are among the smallest cells, approximately 0.3 µm long, and lack a cell wall. Plasmids are small circular DNA molecules outside the bacterial genomic DNA. They can confer properties such as antibiotic resistance and help monitor transformation with foreign DNA.

Ref: NCERT Class 11 Biology Chapter 8: Cell: The Unit of Life Prokaryotic Cell - Structure and Components

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DNA & RNA isolation

What causes the separation of plasmid DNA from chromosomal DNA?

In alkaline lysis for plasmid isolation, covalent closed circular supercoiled plasmid DNA and linear chromosomal DNA behave differently due to topology. Under alkaline conditions with SDS, both are denatured. Upon rapid neutralization, small supercoiled plasmids renature quickly because their two strands remain interlinked topologically, becoming soluble. Large chromosomal fragments cannot correctly reanneal and form an insoluble network with SDS-protein complexes and precipitate. Thus separation is based on topological differences and renaturation kinetics, not merely size variation or charge differences alone.

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.

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RDT Basics

Which enzyme prevents self-ligation of plasmid DNA?

Alkaline phosphatase hydrolyzes 5'-phosphomonoester bonds, converting 5'-phosphate termini of linearized plasmid DNA into 5'-hydroxyl groups. DNA ligase strictly requires a 5'-phosphate and adjacent 3'-hydroxyl to catalyze phosphodiester bond formation, so dephosphorylated vector molecules cannot self-circularize, thereby suppressing empty vector background. In cloning, the foreign insert retains chemically intact 5'-phosphates supplied by restriction digestion or polynucleotide kinase, enabling ligation at each junction to form a heteroduplex with two nicks that are repaired after transformation by bacterial repair machinery, greatly improving recombinant recovery.

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.

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