Skip to content
Login Register

#DpnI

3 public questions tagged with this topic.

Transcript analysis

Which enzyme digests methylated parental DNA in inverse PCR?

Parental plasmids propagated in most Escherichia coli strains are methylated by Dam methyltransferase at N6 position of adenine within sequence GATC. After inverse PCR mutagenesis, mixture contains both methylated wild-type template and unmethylated mutated amplicon. Selective removal of parental background exploits methylation sensitivity. DpnI specifically recognizes GATC only when adenine is methylated, introducing double-strand breaks and fragmenting template into non-functional pieces. Newly amplified mutated DNA lacking methylation resists digestion and remains intact for transformation. This enzymatic selection dramatically improves efficiency, explaining why DpnI treatment is indispensable for site-directed mutagenesis protocols.

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.

View Details
DNA-Protein Interaction analysis

The PCR mix must be treated with DpnI to:

In inverse PCR mutagenesis, reaction mixture contains parental methylated plasmid propagated in Dam positive bacteria and newly synthesized unmethylated mutated copies produced by high-fidelity polymerase in vitro. To enrich mutants, mixture is incubated with DpnI restriction enzyme which recognizes methylated GATC sites present only in parental template, introducing double-strand breaks and fragmenting it. This selective digestion leaves unmethylated mutated amplicons intact, drastically lowering wild-type background upon transformation. Treatment therefore digests template DNA while retaining mutated copies, rather than cutting all DNA or degrading RNA.

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.

View Details
Hybridization

In inverse PCR, DpnI is used to:

In inverse PCR-based site-directed mutagenesis, DpnI endonuclease plays crucial role in selection of newly synthesized mutant plasmids. DpnI specifically recognizes methylated GATC sequences, which occur frequently in parental plasmid DNA propagated in dam+ E. coli strains. Unmethylated PCR product carrying mutation is resistant to DpnI cleavage, whereas methylated parental strands are digested into fragments. This selective removal enriches mutant clones and reduces background wild-type colonies after transformation. DpnI does not cleave mutant DNA, synthesize RNA, or ligate fragments; its function is methylation-dependent restriction for background elimination.

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.

View Details