BenchMasteryMolecular biology techniques & virtual lab

Site-directed mutagenesis

One reaction in which two complementary or partly overlapping mutagenic primers amplify the entire plasmid with a proofreading polymerase. The methylated parental template is then destroyed with DpnI and the nicked circular product is transformed directly, with no ligation and no cloning step.

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QuikChange-style PCR

One reaction in which two complementary or partly overlapping mutagenic primers amplify the entire plasmid with a proofreading polymerase. The methylated parental template is then destroyed with DpnI and the nicked circular product is transformed directly, with no ligation and no cloning step.

How it differs from the standard method

  • Twelve to eighteen cycles is enough: the product plateaus and extra cycles only add polymerase errors across the whole plasmid.
  • DpnI is not optional. Without it the surviving supercoiled parent out-transforms the nicked product and most clones are wild type.
  • Start from 5–50 ng of template; overloading the reaction leaves more parent than a standard one-hour digest can clear.
  • Works for substitutions, and for short insertions and deletions if the primer arms are lengthened.
  • Colony numbers are low by PCR standards (tens, not hundreds) because the product is nicked rather than ligated.
In this lesson · about 5 min read
  1. A primer that deliberately does not match
  2. Amplifying a whole plasmid, then destroying the parent
  3. Why you sequence the whole amplified region
  4. Design tricks worth knowing
  5. The alternative: build it from two halves
  6. Key terms
  7. When to use it
  8. Sub-techniques

1A primer that deliberately does not match

Site-directed mutagenesis works because a primer does not have to match perfectly to anneal. Put the change you want in the middle of an oligonucleotide and leave enough correct sequence on either side, and the two matching arms hold the primer on the template while the mismatched middle is simply copied as the primer wrote it.

2Amplifying a whole plasmid, then destroying the parent

In the most common format the two mutagenic primers point away from each other, or overlap each other, and the polymerase copies the entire plasmid. The product is a full-length circle whose two strands are nicked rather than ligated, and it carries the change.

methylated

Parent

Grown in a Dam⁺ strain, supercoiled, highly transformable. DpnI cuts it to pieces.

unmethylated

Product

Made by the polymerase, nicked, poorly transformable. DpnI ignores it.

ratio

What transforms

The plate is decided by product × transformability against surviving parent. A full digest from 5–50 ng of template puts that ratio above 95%.

3Why you sequence the whole amplified region

The polymerase copied every base of the plasmid, not just the codon you were aiming at, so an unintended substitution can be anywhere: in the promoter, in the tag, in the resistance gene, in a different part of the coding sequence. Even a proofreading enzyme makes mistakes, and errors accumulate with the number of cycles: a few thousandths of an error per molecule over twelve to eighteen cycles with a high-fidelity polymerase, but a large fraction of a mutation per molecule with a non-proofreading enzyme over thirty-five cycles.

4Design tricks worth knowing

Two habits pay for themselves. The first is a silent second change: alter another base in the same codon block so that a nearby restriction site is created or destroyed without changing the protein.

Dam methylation
Methylation of the adenine in GATC by E. coli Dam methyltransferase. Present throughout a plasmid grown in a standard strain, absent from anything synthesised in vitro, which is what makes the parent and the product distinguishable.
DpnI
A restriction enzyme that cuts GATC only when the adenine is Dam-methylated. Unusual among restriction enzymes in requiring methylation rather than being blocked by it.
Effective Tm
The melting temperature of the part of the primer that actually pairs, reduced by the cost of the mismatch or loop. Always below the Tm a calculator gives for the full-length primer assuming a perfect match.

5The alternative: build it from two halves

Overlap extension takes a different route. Two separate reactions amplify the region as two halves, each using one mutagenic primer, so both halves end in the mutated sequence and share a stretch of identical sequence across it.

Key terms

Practise these as flashcards →
Mutagenic primer

An oligonucleotide carrying the intended change in its middle, with enough correct sequence on each side to anneal: about 10–15 nt per side for a substitution, 15–20 for an insertion or deletion.

Dam methylation

Adenine methylation of GATC by the host strain, present in the plasmid you put into the reaction and absent from the product the polymerase makes.

DpnI digestion

The step that destroys the methylated parental plasmid after amplification, so that what transforms is mostly the new, unmethylated product.

Overlap extension

Making the change in two overlapping halves and then joining them through their shared sequence, instead of amplifying the whole plasmid in one reaction.

Silent diagnostic change

A second base change that does not alter the protein but creates or destroys a restriction site, so clones can be screened by digest before sequencing.

Second-site mutation

An unintended polymerase error elsewhere in the amplified DNA. Invisible unless you sequence the whole amplified region.

When to use it

  • Testing what a specific residue does: an active-site mutant, a phosphorylation site, a conserved residue flagged by an alignment.
  • Reproducing a disease-associated variant in an expression construct, or reverting one.
  • Adding or removing a short tag, a linker or a few residues at a defined position.
  • Repairing a construct: correcting a mutation a previous round of cloning introduced, or removing an unwanted restriction site before a digest-based cloning step.

Compared with alternatives

Seamless assembly →

For anything larger than a few residues, building the construct from synthesised fragments with overlaps is usually quicker and cleaner than mutagenising an existing plasmid.

CRISPR-Cas9 editing →

Changes the gene in its own genomic context rather than on a plasmid, which is what you need when expression level and regulation matter.

Base & prime editing →

Makes single-base changes in a genome without a double-strand break, the in-cell counterpart of what mutagenesis does to a plasmid in a tube.

Sanger sequencing →

The readout that decides whether a mutagenesis worked, and the reason the whole amplified region has to be covered rather than just the target codon.

Bacterial transformation →

The step where the parental background shows up: nicked product and surviving supercoiled parent compete for the same competent cells.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

One reaction in which two complementary or partly overlapping mutagenic primers amplify the entire plasmid with a proofreading polymerase. The methylated parental template is then destroyed with DpnI and the nicked circular product is transformed directly, with no ligation and no cloning step.

  • Twelve to eighteen cycles is enough: the product plateaus and extra cycles only add polymerase errors across the whole plasmid.
  • DpnI is not optional. Without it the surviving supercoiled parent out-transforms the nicked product and most clones are wild type.
  • Start from 5–50 ng of template; overloading the reaction leaves more parent than a standard one-hour digest can clear.
  • Works for substitutions, and for short insertions and deletions if the primer arms are lengthened.
  • Colony numbers are low by PCR standards (tens, not hundreds) because the product is nicked rather than ligated.