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A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method
Ali Asghar Karkhane1, Bagher Yakhchali1, Ferdous Rastgar Jazii1
1Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
Iranian Journal of Biotechnology
|September 30, 2017
Summary
A new nested-SOE-PCR method enhances gene mutation generation by increasing product yield and specificity. This improved technique reduces non-specific products, making it more efficient for studying enzyme structure and function.
Area of Science:
- Molecular Biology
- Enzymology
- Biotechnology
Background:
- Splicing by overlap extension (SOE) PCR is a key technique for introducing mutations in enzyme coding sequences.
- Understanding specific residue roles is crucial for analyzing protein structure and function.
Purpose of the Study:
- To introduce a nested-SOE-PCR (N-SOE-PCR) method for enhanced gene mutation generation.
- To improve the specificity and yield of mutations created by SOE-PCR.
Main Methods:
- Genomic DNA from Bacillus thermocatenulatus was extracted.
- Nested PCR was employed to amplify wild-type and mutant Bacillus thermocatenulatus lipase gene variants.
- A three-primer approach in N-SOE-PCR amplifies mutagenic fragments, with subsequent nested and gene-specific primer rounds.
Main Results:
- N-SOE-PCR significantly increased the yield and specificity of amplified products compared to conventional SOE-PCR.
- The improved method demonstrated a substantial reduction in non-specific PCR products.
Conclusions:
- The addition of two primers in N-SOE-PCR enhances method specificity.
- Deeper primer annealing within amplicons improves efficiency and primer attachment.
- Optimized primer positioning boosts binding affinity in the third amplification round.
