Q. 39 DpnI is used to digest the PCR product during site directed mutagenesis because
(A) DpnI digests irrespective of methylation status of DNA
(B) DpnI digests only unmethylated DNA
(C) DpnI digests only methylated DNA
(D) DpnI digests GC-rich sequences
DpnI digests methylated DNA from bacterial templates during site-directed mutagenesis, ensuring only mutated PCR products survive. The correct answer is (C) DpnI digests only methylated DNA.
Correct Answer
Option (C) DpnI digests only methylated DNA is correct. In site-directed mutagenesis protocols, template plasmids from E. coli carry Dam methylation at GATC sequences, making them susceptible to DpnI cleavage. PCR-amplified mutated strands lack methylation and remain intact for transformation.
Options Breakdown
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(A) DpnI digests irrespective of methylation status of DNA: Incorrect, as DpnI specifically requires adenine methylation in GATC for cleavage; unmethylated DNA resists digestion.
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(B) DpnI digests only unmethylated DNA: Incorrect; this reverses the enzyme’s function, which targets methylated parental templates to enrich unmethylated mutants.
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(C) DpnI digests only methylated DNA: Correct; it selectively removes methylated template, boosting mutation efficiency to near 100% in protocols.
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(D) DpnI digests GC-rich sequences: Incorrect; specificity ties to methylation, not GC content, distinguishing it from sequence-only nucleases.
Protocol Role
DpnI treatment follows PCR amplification in kits like QuikChange, incubating at 37°C for 15-60 minutes to destroy template. Unmethylated nicked plasmids transform efficiently into E. coli, minimizing wild-type background. This step proves vital for precise genetic engineering in molecular biology labs.
