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Figure 1 | BMC Biotechnology

Figure 1

From: MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR

Figure 1

The MALS mutagenesis procedure. Target DNA is individually amplified with two pairs of oligonucleotides SO1/IR1 and SO2/IF1. IF1 and IR1 are phosphorylated (rose circles), while SO1 and SO2 are dephosphorylated. The desired mutation is incorporated at the 5' end of IR1 oligonucleotide (shown in red). Panel 1 on the right shows the types of mutations available with MALS. PCR-generated DNA fragments are ligated. The resulting DNA population consists of homomeric ligation products (type A), non-ligated DNA fragments (type B), and molecules representing full length target DNA (type C) (panel 2 on the right). The entire DNA population is then amplified with suppression oligonucleotides SO1 and SO2. Intramolecular hybridization of inverted repeat sequences prevents efficient replication of type A molecules, while type B molecules amplify linearly (panel 3 on the right). Only heteromeric ligation products (type C) amplify exponentially. The resulting DNA population predominantly consists of type C molecules. An aliquot of the PCR mixture is used for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA. Finally, SO1 and SO2 suppression sequences (green and blue, respectively) are digested with restriction endonucleases and DNA fragments are ligated with linearized vector for subsequent subcloning.

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