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Methods For Site-Directed Mutagenesis
Methods For Site-Directed Mutagenesis
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Figure 1. Site-Directed Mutagenesis by Traditional PCR. Primers incorporating the desired base changes are used in PCR. As the primers are extended, the mutation is created in the resulting amplicon.
Primer Extension Site-directed mutagenesis by primer extension involves incorporating mutagenic primers in independent, nested PCRs before combining them in the final product [2]. The reaction requires flanking primers (A and D) complementary to the ends of the target sequence, and two internal primers with complementary ends (B and C). These internal primers contain the desired mutation and will hybridize to the region to be altered. During the first round of PCR, the AB and CD fragments are created. These products are mixed for the second round of PCR using primers A and D. The complementary ends of the products hybridize in this second PCR to create the final product, AD, which contains the mutated internal sequence (Figure 2A). Longer insertions can be incorporated by using especially long primers, such as IDT Ultramer oligonucleotides. To create a deletion, the internal primers, B and C, are positioned at either side of the region to be deleted to prevent it from being incorporated within fragments AB and CD from the first round of PCR. The complementary sequences at the ends of the these fragments, created by primers B and C, enable hybridization of AB to CD during the second round of PCR, and the final product with the desired deletion (AD)
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is created (Figure 2B).
Figure 2. Site-Directed Mutagenesis by Prim er Extension. (A) Insertion: Primers B and C contain the complementary sequence that w ill be inserted (blue line). Tw o reactions are performed in the first round of PCR using primer pairs A/B (1) and C/D (2). The resulting amplicons are mixed w ith primer pair A/D for the second round of PCR. The complementary ends of the first round amplicons hybridize and the PCR creates the final product w ith the desired insertion. (B) Deletion: Primers B and C are located on either side of the sequence to be deleted, and contain sequence from both sides of the deletion (black or gray additions that match the black or gray original sequence). Tw o reactions are performed for the first round of PCR using primer pairs A/B and C/D. The amplicons are mixed w ith primer pair A/D for the second round of PCR. The overlapping regions of these amplicons hybridize and the PCR creates the final product w ith the desired deletion.
Inverse PCR Inverse PCR enables amplification of a region of unknown sequence using primers oriented in the reverse direction [3]. An adaptation of this method can be used to introduce mutations in previously cloned sequences. Using primers incorporating the desired change, an entire circular plasmid is amplified to delete (Figure 3A), change (Figure 3B), or insert (Figure 3C) the desired sequence.
Figure 3. Site-Directed Mutagenesis by Inverse PCR. The primers used are 5phosphorylated to allow ligation of the amplicon ends after PCR. A high fidelity DNA polymerase that creates blunt-ended products is used for the PCR to produce a linearized fragment w ith the desired mutation, w hich is then recircularized by intramolecular ligation. (A) Deletion: Primers that hybridize to regions on either side of the area to be deleted are used. (B) Substitution: One of the primers contains the desired mutation (blue bubble). (C) Insertion: The primers hybridize to regions on either side of the location of the desired insertion (black, dotted line). One primer contains the additional sequence that w ill be inserted (blue line).
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References 1. Zoller MJ (1991) New molecular biology methods for protein engineering. Curr Opin Biotechnol, 2(4): 526531. 2. Reikofski J and Tao BY (1992) Polymerase chain reaction (PCR) techniques for site-directed mutagenesis. Biotechnol Adv, 10(4): 535547. 3. Ho SN, Hunt HD, Horton RM, et al. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77(1):5159. 4. Ochman H, Gerber AS, and Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics, 120(3): 621623. Authors: Jaime Sab el and Nicola Brookman-Amissah are Scientific Writers at IDT. Related Articles Using Site-directed Mutagenesis to Elucidate Structure: Function Relationships Oligonucleotide Quality Requirements for Mutagenesis Protocols Mutagenesis Made Easy with Ultramer Oligos Mutagenesis Application Guide: Experimental Overview, Protocol, Troubleshooting
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