Techniques For Detection Of Known Mutations And Sequence Variants

Restriction endonucleases have proven to be extremely useful in the analysis and characterization of PCR products. Digestion of PCR products with endonucleases can be used to confirm the amplification of desired sequences when the size of the fragments can be predicted from known restriction sites. In addition, it can be used to identify sequence variants and provide linkage information for pedigree analysis when a mutation has not been identified or before a gene is cloned (52,53).

Restriction endonucleases protect bacteria from invasion by foreign DNA by recognizing and cleaving specific sequences in double-stranded DNA (54). The bacteria's own DNA is protected from digestion through methylation or modification of the restriction sites; thus, they are not recognized by the enzyme. There are three classes of endonuclease with different cofactor requirements and different DNA recognition abilities. The class II enzymes are most commonly used in molecular biology applications. They require only the presence of Mg2+, recognize DNA sequences approx 4-8 bases long and cleave at, or near, the recognition site. Many of these sites are palin-dromic, and when cleaved, they result in "blunt" or "sticky" ends. These new ends are extremely advantageous for ligation of the fragment into vectors for further manipulation. The appropriate digestion conditions and buffer for each enzyme are usually supplied by the manufacturer. Because most enzymes are active at 37°C, several can be combined in one reaction if a single product is to be cut at several sites or if a multiplex reaction contains several different products with different restriction sites. The resulting fragments are separated on an agarose or acrylamide gel, depending on the required resolution and stained with ethidium bromide for visualization.

There are numerous restriction sites throughout any region of DNA. Some of these sites are polymorphic in that on a given allele, the site might be present or absent. This could be part of normal variation and not result in any disease. The presence or absence of the site affects whether the DNA fragment is cleaved by an endonuclease. If the polymorphism is closely linked to a disease locus, in some families it could be used as a marker to follow inheritance of the mutant (disease-producing) allele. Prior to the development of direct detection methods to identify cystic fibrosis mutations, analysis of linked restriction fragment length polymorphisms (RFLPs) was used for prenatal diagnosis (53) and is still useful when both mutations in the parents have not been determined. This type of analysis requires that an affected individual and both parents be available for testing to ascertain which parental alleles carry the mutant genes. DNA obtained from chorionic villus sampling or amniocentesis is the template for PCR amplification of the region encompassing the polymorphism. Based on the restriction fragment patterns resulting from digestion of the PCR products, the genotype of the fetus is determined. Linkage marker analysis for prenatal diagnosis is not 100% accurate because of the possibility of recombination between the alleles during meiosis. Therefore, it is desirable to analyze several markers to increase the certainty of the diagnosis.

Once a disease gene is identified and mutations have been detected and characterized, RFLP analysis might be used to screen samples for the same mutations if they create/destroy a restriction site. The sequence flanking the mutation is amplified and digested with the appropriate enzyme, and the fragments are resolved by gel electrophoresis. For example, the cystic fibrosis mutation 2789 + 5G > A creates a SspI cutting site. When a 305-bp region of the gene that encompasses this mutation is amplified and digested, three fragments result. (One additional fragment results from a constitutive site present in both alleles.) The wild-type allele is cut only once at the constitutive site to produce two fragments (Fig. 16) (55). Mutations and sequence variants that alter restriction sites have been identified in virtually all genes studied.

Unfortunately, a sequence variant does not always change a restriction site. Therefore, in order to preserve the simplicity of mutation detection by PCR/restriction digestion,

PCR-mediated site-directed mutagenesis can be applied (56). This technique creates or destroys restriction sites in the PCR product by introduction of a base substitution near the mutation by modifying the primers. This allows the detection of point mutations as well as small insertions and deletions that cannot be resolved through gel fractionation of the PCR products. A polymerase must be used that does not have exonucle-ase activity, or the mismatched primer will be corrected. The mismatched base can be several bases from the 3' end of the primer to stabilize the primer-template hybrid without decreasing the efficiency of the polymerase or the specificity of the amplification. Several cystic fibrosis mutations can be detected

Microsatellite Instability
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