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Fig. 16. Restriction fragment length polymorphism analysis of the cystic fibrosis mutation 2789+5G>A. (A) The wild-type PCR product is digested with Sspl to two fragments resulting from the presence of a constitutive restriction site not associated with the mutation. When the G (surrounded by a small box) is mutated to an A, an additional Sspl site is created to yield three fragments. The Sspl site is indicated by the shaded box and the cutting site is indicated by the arrows. (B) A 305-bp region of the CFTR gene that flanks the 2789+5G>A mutation was amplified and digested with Sspl. The PCR products were separated by electrophoresis through a 4% agarose gel and then stained with ethidum bromide. Lane 1: ^X174 DNA size markers; lane 2, individual heterozygous for the 2789+5G>A mutation (265- and 217- bp fragments); lane 3, normal individual (265bp fragment); lane 4, water blank. The 40- and 48-bp fragments migrate quickly through the gel and are not visible. (Courtesy of Michelle L. Blalock, Molecular Genetics Laboratory, University of North Carolina Hospitals.)

by introduced restriction sites with mismatches 1,2 or 3 bases from the 3' end (57,58). An example of an assay for the G542X mutation is shown in Fig.17.

Failure of the endonuclease to digest a PCR product can lead to misleading results in RFLP or PCR-mediated site-directed mutagenesis assays. Control samples, homozygous and heterozygous for both alleles, should be amplified and digested at the same time as the unknown. Additional endogenous or engineered restriction sites within the PCR product can act as an internal control for complete digestion. In some instances, such as the G542X and 2789 + 5G > A cystic fibrosis mutations, a constitutive site within the PCR product is valuable as an internal control for digestion (57). In cases in which a constitutive site is not found on the amplicon being investigated, coamplifi-cation of a sequence containing the appropriate restriction site in an irrelevant gene for use as a restriction digest control is highly recommended.

The decision to introduce a restriction site into either allele is usually based on the location of the mismatch from the 3' end of the primer and the commercial availability of the enzyme. However, the assay is more specific for a particular mutation when the primers are designed to introduce a cutting site into the mutant allele rather than ablate an existing one (57,59). The reason for this is that if the restriction site is removed in the mutant allele, a rare polymorphism in the restriction site can also prevent the digestion of the PCR product, giving a false-positive result.

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