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Biochemistry

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Purines, Pyrimidines and Nucleic Acid Metabolism

Question
40 out of 59
 

Oligomer with a mutant base substitution is used as a primer: (AIIMS May 2009)



A Site directed mutagenesis

B Error based PCR with mutagenesis

C Transfection

D RFLP

Ans. A Site directed mutagenesis

Site-directed mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. In general, site-directed mutagenesis requires that the wild-type gene sequence be known.

This technique is also known as site-specific mutagenesis or Oligonucleotide-directed mutagenesis .

Basic steps in a site-directed mutagenesis method.

a. Cloning the DNA of interest into a plasmid vector.

b. The plasmid DNA is denatured to produce single strands.

c. A synthetic oligonucleotide with desired mutation (point mutation, deletion, or insertion) is annealed to the target region.

d. Extending the mutant oligonucleotide using a plasmid DNA strand as the template.

e. The heteroduplex is propagated by transformation in E. coli.

After propagation, in theory, about 50% of the produced heteroduplex will be mutants and the other 50% will be the "wild type" (no mutation). In commercial mutagenesis kits, some selection and enrichment methods have been used to favor the production of mutants.

Purines, Pyrimidines and Nucleic Acid Metabolism Flashcard List

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