Cassette mutagenesis is a type of

site-directed mutagenesis Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...

that uses a short, double-stranded oligonucleotide sequence (

gene cassette In biology, a gene cassette is a type of mobile genetic element that contains a gene and a recombination site. Each cassette usually contains a single gene and tends to be very small; on the order of 500–1000 base pairs. They may exist incorpo ...

) to replace a fragment of target DNA. It uses complementary restriction enzyme digest ends on the target DNA and

to achieve specificity. It is different from methods that use single

oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...

in that a single gene cassette can contain multiple mutations. Unlike many site directed mutagenesis methods, cassette mutagenesis also does not involve primer extension by

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...

Mechanism

First,

restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class ...

s are used to cleave near the target sequence on DNA contained in a suitable

vector Vector most often refers to: *Euclidean vector, a quantity with a magnitude and a direction *Vector (epidemiology), an agent that carries and transmits an infectious pathogen into another living organism Vector may also refer to: Mathematic ...

. This step removes the target sequence and everything between the restriction sites. Then, the synthetic double stranded DNA containing the desired mutation and

ends End, END, Ending, or variation, may refer to: End *In mathematics: **End (category theory) ** End (topology) ** End (graph theory) ** End (group theory) (a subcase of the previous) ** End (endomorphism) *In sports and games **End (gridiron footba ...

that are complementary to the restriction digest ends are ligated in place of the sequence removed. Finally, the resultant

construct Construct, Constructs or constructs may refer to: * Construct (information technology), a collection of logic components forming an interactive agent or environment ** Language construct * ''Construct'' (album), a 2013 album by Dark Tranquillity ...

sequenced In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succi ...

to check that the target sequence contains the intended mutation.

Usage

The use of synthetic gene cassette allows total control over the type of mutation that can be generated. When studying protein functions, cassette mutagenesis can allow a scientist to change individual

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...

s by introducing different

codon The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...

s or omitting codons. By including the SD sequence and the first few codons of a gene, a scientist can easily and dramatically affect the expression level of a protein by altering these

regulatory sequence A regulatory sequence is a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes within an organism. Regulation of gene expression is an essential feature of all living organisms and vi ...

Limitations

To use this method, the sequence of the target sequence and nearby restriction sites must be known. Since

s are used, for this method to be useful, the restriction sites flanking the target DNA has to be unique in the

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

system so that the

can be inserted with specificity. The length of the sequence flanked by the restriction sites is also a limiting factor due to the use of synthetic gene cassettes.

Advantages

Since one gene cassette can contain multiple mutations, less total oligonucleotide synthesis and purification is needed. Compared to mutagenesis methods that requires the synthesis of double stranded DNA using a single stranded template (1-30% in vitro in M13), the efficiency of the ligation of oligodeoxynucleotide cassette is close to 100%. The high efficiency of the mutagenesis means mutants can be screened directly by sequencing. Once the vector is set up with flanking restriction sites, all manipulations (i.e.,

mutagenesis Mutagenesis () is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using l ...

sequencing In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succi ...

expression Expression may refer to: Linguistics * Expression (linguistics), a word, phrase, or sentence * Fixed expression, a form of words with a specific meaning * Idiom, a type of fixed expression * Metaphorical expression, a particular word, phrase, ...

) can be performed in the same

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; howev ...

References

{{Reflist, 30em Genetics techniques Molecular genetics Mutagenesis Protein engineering