Site-Directed Mutagenesis

Advantages

• Both primers contain the mutation.
• In vitro removal of methylated DNA (parental template).
• Simple 3-step workflow***.

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Disadvantages

• Long amplification protocol – 25 cycles (between 4 and 8 hours).
• Low yield amplification of DNA.
• Complementary primers anneal together.
• Tandem primer insertion at or near the mutated site often occurs.
• Large insertions are problematic.
• Large deletions are problematic.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Advantages

• Semi-exponential amplification PCR.
• Both primers contain the mutation.
• In vitro digestion and in vivo degradation of non-mutated parental DNA.
• 95% of clones contain the desired mutation.
• Suitable for insertions of ∼ 45 bases.
• Suitable for large deletions.
• Easy 3-step workflow***.

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Disadvantages

• Long PCR protocol – 25 cycles (between 4 and 8 hours).
• Partially complementary primers may anneal together.
• Tandem primer insertion at or near the mutated site may occur.
• Large deletions may be challenging because of the partial overlap between primers.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Advantages

• High yield exponential PCR.
• Simple primer design.
• In vitro digestion of the template DNA.
• 90% of clones contain the desired mutation.
• Suitable for insertions of ∼ 80 bases or longer if using oligos > 60-mers.
• Ideal for creating large deletions.
• Easy 3-step workflow***.

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Disadvantages

• Long PCR protocol – 25 cycles (between 4 and 8 hours or 1 to 2 hours using Fast & Steep PCR).
• Only 1 primer contains the mutation which may generate non-methylated and non-mutated PCR products.
• Primers or Dpn I-generated fragments are likely to be inserted at the ligation site.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Advantages

• High yield exponential PCR***.
• Versatile technique.
• In vitro digestion of the template DNA (optional).
• 90% of clones contain the desired mutation and will be intact.
• Suitable for large insertions.
• Suitable for large deletions.
• Ideal for creating multiple mutations.
• 4-step workflow (gel extraction of PCR amplicons is recommended)

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Disadvantages

• Multiple primers to order (minimum of 4 primers for creating 1 mutation when performing whole vector amplification)
• Multiple PCR reactions to perform.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification requires subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Clear benefit :

Cleanest way to perform site-directed mutagenesis without altering the backbone vector.

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Advantages

• Both primers at the mutation site contain the desired mutation.
• Short and easy PCR reactions.
• In vitro removal of methylated template DNA may be performed (proceed with caution by verifying restriction sites first)
• Suitable for large insertions
• Suitable for large deletions
• Ideal for introducing multiple mutations (close or distant).
• No subsequent subcloning required and no need to sequence the whole vector.

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Disadvantages

• Multiple primers to order (minimum of 4 primers for creating 1 mutation).
• Multiple PCR reactions to perform.
• Gel extraction and/or PCR purification required.
• Multiple steps to perform, but the vector backbone remains intact through the entire process.

The Fast & Clean Site-Directed Mutagenesis Protocol uses a combination of techniques to achieve unprecedented precision, efficiency and clean site-directed mutagenesis.

• Fast & Steep PCR
• Seamless DNA Assembly Site-Directed Mutagenesis
• 2SPO extension (optional)

Clear benefit :

• Cleanest way to perform site-directed mutagenesis without altering the backbone vector.
• Huge Huge Huge time-saver

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Advantages

• Reduces the number of PCR cycles to a minimum (5-10) (total average run time is 25 min);
• Enables to perform large insertions;
• Enables to perform large deletions;
• Reduces the time normally required to perform site-directed mutagenesis using two-sided PCRs and/or overlap extension PCR;
• Enables at least 3 alternative solutions in case of failure (using the same ssDNA primers);
• Offers a clean alternative to whole vector amplification techniques that require subsequent subcloning steps or sequencing of the entire plasmid. No sublcloning is required.

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Disadvantages

• Multiple primers to order (minimum of 4 primers for creating 1 mutation instead of 2).
• Multiple PCR reactions to perform (but they are completed 4-times faster).
• Gel extraction and/or PCR purification steps required for all purification steps.

Advantages of the Fast & Steep PCR Protocol

Very Fast : 15-45 min for 300 bp and 10 kb respectively.

• PCR cycling time varies depending on template lenght and ramping rates

Efficient : incorporates up to 100% of primers in a very short amount of time.

• GC content and primer specificity may affect the efficiency.

Non-Ambiguious results : PCR amplicons have fully incorporated your primers.

• When performing site-directed mutagenesis, so far, our success rate is 100%.

Low error rate / High-Fidelity : 5 to 10 PCR cycles is sufficient.

• Limits the chances of introducing errors by up to 2E15 -fold (10 vs 25 PCR cycles).