Alkaptonuria is a human autosomal recessive disorder caused by mutation of the HAO gene that encodes the enzyme homogentisic acid oxidase. A map of the HAO gene region reveals four BamHI restriction sites (B1 to B4) in the wild-type allele and three BamHI restriction sites in the mutant allele. BamHI utilizes the restriction sequence 5′-GGATCC-3′. The BamHI restriction sequence identified as B3 is altered to 5′-GGAACC-3′ in the mutant allele. The mutation results in a Ser-to-Thr missense mutation. Restriction maps of the two alleles are shown below, and the binding sites of two molecular probes (probe A and probe B) are identified. DNA samples taken from a mother (M), father (F), and two children (C1 and C2) are analyzed by Southern blotting of BamHI-digested DNA. The gel electrophoresis results are illustrated. Explain how the DNA sequence change results in a Ser-to-Thr missense mutation.

Key Concepts

Missense Mutation

A missense mutation is a type of point mutation where a single nucleotide change results in the substitution of one amino acid for another in the protein sequence. This can affect the protein's function, depending on the properties of the amino acids involved. In the case of alkaptonuria, the mutation changes a serine (Ser) to a threonine (Thr), which may alter the enzyme's activity or stability.

Restriction Enzymes and Sites

Restriction enzymes, like BamHI, are proteins that cut DNA at specific sequences, known as restriction sites. The recognition sequence for BamHI is 5′-GGATCC-3′, and any alteration in this sequence can prevent the enzyme from binding and cutting the DNA. In the mutant allele of the HAO gene, the B3 site is altered to 5′-GGAACC-3′, which means BamHI can no longer recognize or cut at this site, impacting the analysis of the gene.

Southern Blotting

Southern blotting is a technique used to detect specific DNA sequences within a complex mixture. It involves the digestion of DNA with restriction enzymes, followed by gel electrophoresis to separate the fragments, and then transferring the DNA to a membrane for hybridization with labeled probes. This method allows researchers to visualize the presence or absence of specific alleles, such as the wild-type and mutant HAO alleles in the family analysis.