#plasmid DNA

Vaccine

DNA vaccines deliver genes in the form of:

DNA vaccines deliver genetic instructions as circular plasmid DNA rather than protein antigen or RNA. Plasmid constructs typically contain bacterial origin pUC, antibiotic resistance for selection during manufacturing, and eukaryotic expression cassette comprising strong promoter cytomegalovirus CMV immediate early with intron A enhancing transcription, codon optimized antigen gene, polyadenylation signal bovine growth hormone BGH or SV40 preventing mRNA degradation. Plasmids produced in Escherichia coli fermentation high copy number, purified supercoiled isoform removing endotoxin via chromatography. Upon intramuscular delivery with electroporation needles delivering brief electrical pulses increasing membrane permeability or needle free jet injector, plasmid enters myocytes and antigen presenting cells, translocates nucleus independently of cell division, transcribed by RNA polymerase II into mRNA exported then translated. Resulting antigen triggers both humoral and cellular immunity. Advantages include exceptional thermostability stable at room temperature months facilitating transport without stringent cold chain, simple large scale manufacturing, lack of anti-vector immunity permitting homologous boosting, ability to encode multiple antigens single plasmid. Limitations include relatively low transfection efficiency in humans requiring devices, theoretical rare integration at frequencies less than 10^-7 per plasmid. World's first approved ZyCoV-D COVID DNA vaccine India uses three dose regimen.

Ref: Wolff et al. Science 1990 DNA vaccines plasmid; Plotkin DNA vaccines; WHO DNA plasmid.

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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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