Task N.7: site-directed mutagenesis


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overlap extention

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Modified: 7 October 2009, 10:04 AM   User: Gnoni Alessandra  → 

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PCR-mediated overlap extension can create specific nucleotide mutations or generate chimeric gene products.


Figure a illustrates....

1. una volta scelto il nucleotide da modificare usiamo i primers c e b che contengono il nucleotide diverso da inserire.

2. si conducono 2 reazioni di PCR in parallelo: una con i primers a e b e una con i primers c e d

3. mescolando i prodotti di reazione, tra i prodotti del reannealig, ci sarà il segmento AB che si appaia con CD nella zona mutata.

$. La PCR estende a partire dall'estremità 3' e con i primers a e d si ottiene il segmento AD contenente il nucleotide mutato 

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Figure b illustrates.......



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italiano


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overlap extention

From the following article: Gene splicing and mutagenesis by PCR-driven overlap extension Karin L Heckman & Larry R Pease Nature Protocols 2, 924 - 932 (2007)