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#PCR techniques

2 public questions tagged with this topic.

PCR

Radioactive primer labelling is best done with

Radioactive labeling of primers for detection in Sanger sequencing, footprinting, or probe tracking is best achieved by transferring γ-phosphate of [γ-32P]ATP to 5' hydroxyl terminus of oligonucleotide using T4 polynucleotide kinase, yielding 5' 32P-labeled primer. This end-label does not interfere with polymerase extension from 3' end. Alpha-32P labeled deoxynucleotides incorporate internally via polymerase synthesis but not suitable for pre-synthesized primer labeling. Beta and generic notations are not used. Five-prime labeling provides high specific activity enabling autoradiographic detection with superior sensitivity.

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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