#inverse PCR

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.

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Transcript analysis

Inverse PCR mutagenesis includes:

Inverse polymerase chain reaction based mutagenesis allows mutation of entire plasmid without subcloning. Divergent primers containing desired change amplify around circular template generating linear mutated product. Reaction mixture retains original non-mutated parental plasmid isolated from methylation-competent Escherichia coli, which could transform and cause background wild-type colonies. DpnI is a restriction endonuclease that recognizes methylated and hemimethylated sequence 5 prime GATC methylated at adenine by Dam methylase. It cleaves parental methylated DNA into fragments unable to replicate, enriching for newly synthesized unmethylated mutated plasmid, increasing mutagenesis efficiency and reducing false positives.

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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DNA-Protein Interaction analysis

Which statement is true about DpnI enzyme in inverse PCR mutagenesis?

DpnI is methylation-dependent restriction enzyme recognizing tetranucleotide sequence 5'-GATC-3' only when adenine is methylated at N6 position by bacterial Dam methylase. Plasmid DNA isolated from standard laboratory Escherichia coli strains such as DH5alpha or XL1-Blue is fully methylated at GATC sites, rendering it sensitive to DpnI cleavage. In site-directed mutagenesis, newly synthesized strands generated by proofreading polymerase in vitro lack methylation and resist digestion. DpnI treatment selectively destroys parental template, dramatically reducing wild-type background colonies and enriching desired mutants, with no activity on 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.

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DNA-Protein Interaction analysis

What is the correct order in site-directed mutagenesis using inverse PCR?

Inverse PCR based site-directed mutagenesis introduces predetermined nucleotide changes using complementary mutagenic primers. Entire circular plasmid is amplified with high-fidelity polymerase using primers carrying desired substitution, generating linear full-length copies containing mutation. Parental plasmid propagated in Dam methylase positive Escherichia coli contains methylated GATC sequences, while PCR product remains unmethylated. Reaction is digested with methylation sensitive DpnI to fragment parental template, leaving mutated amplicons intact. Digested product is transformed into competent cells where it recircularizes. Simplified workflow follows PCR, then DpnI digestion, then transformation.

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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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