Site-Directed Mutagenesis by Overlap Extension Using the Polymerase Chain Reaction (CAT#: STEM-MT-0039-LGZ)

Introduction

Using this site-directed mutagenesis technique, three variants of the mouse major histocompatibility complex class I gene have been generated, cloned and analyzed. Screening of mutant clones showed at least 98% mutagenesis efficiency. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard site-directed mutagenesis because it is faster, simpler, and nearly 100% efficient in generating mutant products.

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Principle Applications Procedure Materials Notes Related Products

Principle

Overlap extension is a new genetic engineering method. Complementary oligodeoxyribonucleotide (oligo) primers and polymerase chain reaction are used to generate DNA fragments with overlapping ends. These fragments join together in a subsequent "fusion" reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to act as a primer for the 3' extension of the complementary strand. The resulting fusion products were further amplified by PCR. Specific changes in nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into overlapping oligonucleotide primers.

Applications

Genetic Engineering

Procedure

1. Primer design.
2. Preparation of reaction system.
3. Set up the program.

Materials

Sample: depends on the customer's analysis requirements

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