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How does QuikChange mutagenesis work?

How does QuikChange mutagenesis work?

QuikChange™ works by using a pair of complementary primers with a mutation. In a round of PCR cycles these primers anneal to the template DNA, replicating the plasmid DNA with the mutation. The mutant DNA product has a strand break (nick) (Figure ​ 1A).

What PCR is used for point mutation?

Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s).

What is the purpose of site-directed mutagenesis?

Site-directed mutagenesis (SDM) methods are used to generate cloned DNAs with modified sequences for examining the importance of specific residues in protein structure and function. SDM represents the primary rational method in protein engineering and for altering enzyme substrate selectivity [1, 2].

Can you detect a point mutation by PCR?

The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3′-end of the ddNTP-blocked primer.

What is the difference between random and site-directed mutagenesis?

Random mutagenesis is the process of introducing mutations randomly while Site Directed mutagenesis is the process in which mutations are introduced in a site-specific manner to specific locations in the DNA or to specific nucleotides. This is the difference between Random mutagenesis and Site Directed mutagenesis.

How do you design a point mutation primer?

Primers should be between 25 and 45 bases in length, with a melting temperature (Tm) of ≥78°C. The desired mutation (deletion or insertion) should be in the middle of the primer with ~10–15 bases of correct sequence on both sides and minimum GC content of 40% and should terminate in one or more C or G bases.

What is tail PCR?

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is a fast and efficient method to amplify unknown sequences adjacent to known insertion sites in Arabidopsis.

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