We’ve updated our DECODED article library!

Get answers to your research questions, with articles sorted by application. Try it now »

Core Concepts
Scientific Fundamentals Explained

Methods for site-directed mutagenesis

Site-directed mutagenesis is an in vitro method for creating a specific mutation in a known sequence. While often performed using PCR-based methods, the availability of custom-designed, synthetic, double-stranded DNA (dsDNA) fragments can drastically reduce the time and steps required to obtain the same  sequence changes.

In this article we describe several PCR-based methods for site-directed mutagenesis. Primers designed with mutations can introduce small sequence changes, and primer extension or inverse PCR can be used to achieve longer mutant regions. Using these site-directed mutagenesis techniques allows researchers to investigate the impact of sequence changes or screen a variety of mutants to determine the optimal sequence for addressing the question at hand. The IDT Mutagenesis Application Guide provides more details on these approaches.

Read our follow-up article, Site-directed mutagenesis—improvements to established methods, to learn how to use a simplified, alternative approach for generating similar mutagenesis designs quickly, with custom-designed, dsDNA fragments.

Traditional PCR

When PCR is used for site-directed mutagenesis, the primers are designed to include the desired change, which could be base substitution, addition, or deletion (Figure 1). During PCR, the mutation is incorporated into the amplicon, replacing the original sequence.

Mutations introduced by PCR can only be incorporated into regions of sequence complementary to the primers and not regions between the primers [1].


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) is created (Figure 2B).

Figure 2. Site-directed mutagenesis by primer extension
. (A) Insertion: Primers B and C contain the complementary sequence that will be inserted (blue line). Two reactions are performed in the first round of PCR using primer pairs A/B (1) and C/D (2). The resulting amplicons are mixed with 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 with 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). Two reactions are performed for the first round of PCR using primer pairs A/B and C/D. The amplicons are mixed with primer pair A/D for the second round of PCR. The overlapping regions of these amplicons hybridize and the PCR creates the final product with 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 5’-phosphorylated 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 with the desired mutation, which 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 will be inserted (blue line).


  1. Zoller MJ (1991) New molecular biology methods for protein engineering. Curr Opin Biotechnol, 2(4): 526–531.
  2. Reikofski J, Tao BY (1992) Polymerase chain reaction (PCR) techniques for site-directed mutagenesis. Biotechnol Adv, 10(4): 535–547.
  3. Ho SN, Hunt HD, et al. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77(1):51–59.
  4. Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics, 120(3): 621–623.

Product focus: Reagents for mutagenesis

Custom double-stranded DNA—gBlocks® Gene Fragments

These double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, are designed by you, and are shipped in 2–5 working days for affordable and easy gene construction or modification. They have been used in a wide range of applications including mutagenesis, CRISPR-mediated genome editing, antibody research, codon optimization, and aptamer expression. They can also be used for generating qPCR standards. 

gBlocks Gene Fragments are also available as dsDNA fragment pools (gBlocks Gene Fragments Libraries) that contain up to 18 consecutive variable bases (N or K) for recombinant antibody generation or protein engineering.

Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.


IDT offers custom DNA synthesis on scales from 25 nmole to 10 μmole, and beyond. Every oligonucleotide primer is deprotected and desalted to remove small molecule impurities. Oligonucleotides are quantified twice by UV spectrophotometry to provide an accurate measure of yield and are quality control checked by mass spectrometry.

Same Day Custom Oligos

Ultramer™ Oligonucleotides

Ultramer Oligonucleotides are 25–200 bases long and are synthesized using IDT proprietary, high-fidelity synthesis systems and chemistries. They are the longest, highest-quality oligonucleotides commercially available and are ideal for demanding applications like cloning, ddRNAi, and gene construction. Researchers can save a great deal of time and trouble in these applications through direct synthesis of the entire target fragment. Ultramer Oligonucleotides are available on several scales, and can come with attached modifications such as 5′ phosphate, biotin, and amino modifiers C6 and C12. Internal degenerate bases, as well as deoxyuracil and deoxyInosine modifications are also available.

Learn more about Ultramer Oligos

Phosphate modifications

Phosphate modifications may be added to any primer or Ultramer Oligonucleotide. 5′ phosphorylation is necessary if the product will be used as a substrate for DNA ligase, as when two pieces will be ligated together to create a combined, longer product. 3′ phosphorylation will inhibit degradation by some 3′-exonucleases and can be used to block extension by many DNA polymerases.


IDT provides a confidential custom gene synthesis service. By ordering genes from IDT, researchers not only save money spent on reagents necessary for construction, cloning, and sequencing, but can also save time by outsourcing the manufacturing of hard-to-clone gene sequences which often results in repeated failures. At IDT, all genes are constructed using Ultramer Oligonucleotides and the highest fidelity next generation synthesis technology available. Genes arrive in a plasmid cloning vector and are ready for use in a variety of applications.

Learn more about Custom Gene Synthesis

Related articles

Site-directed mutagenesis—improvements to established methods—Site-directed mutagenesis techniques have relied primarily on PCR and standard cloning methods. Read about some of the common cloning methods used for mutagenesis and how double-stranded DNA fragments (gBlocks Gene Fragments) can save you both time and money.

Mutagenesis using gBlocks® Gene Fragments—Citation summary: Learn how just 3 synthetic, high fidelity, double-stranded gBlocks Gene Fragments were used to mutate 18 different sites over the entire exon 7, 1039 bp sequence.

Using Site-directed Mutagenesis to Elucidate Structure: Function Relationship—Read how this research group uses site-directed mutagenesis to identify critical small RNA binding regions and transcription factor regulation in E. coli.

Oligonucleotide Quality Requirements for Mutagenesis Protocols—Learn about the importance of oligo purity and how IDT tests its oligos to ensure they meet these standards.

Mutagenesis Made Easy with Ultramer Oligos—Ultramer primers are oligos that range in length from 25–200 bases. See how these long primers can be especially useful to you in PCR and site-directed mutagenesis reactions.

Mutagenesis Application Guide: Experimental Overview, Protocol, Troubleshooting—Get this detailed guide on traditional in vitro mutagenesis methods that use long oligonucleotides and PCR to make sequence changes.

Authors: Jaime Sabel is a scientific writer, and Nicola Brookman-Amissah, PhD, is senior scientific writer at IDT.

© 2012, 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.