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.

In a separate figure, draw the gel electrophoresis band patterns for all the genotypes that could be found in children of this couple.

Verified step by step guidance

1. Step 1: Understand the genetic basis of the problem. Alkaptonuria is an autosomal recessive disorder caused by a mutation in the HAO gene. The mutation alters a BamHI restriction site, changing the sequence from 5′-GGATCC-3′ to 5′-GGAACC-3′, which results in a Ser-to-Thr missense mutation. This change affects the restriction map of the mutant allele.
2. Step 2: Analyze the restriction maps provided for the wild-type and mutant alleles. The wild-type allele has four BamHI restriction sites (B1 to B4), while the mutant allele has three BamHI restriction sites due to the alteration at B3. This difference will result in distinct fragment sizes when the DNA is digested with BamHI.
3. Step 3: Review the Southern blotting technique. Southern blotting involves digesting DNA with a restriction enzyme (in this case, BamHI), separating the fragments by gel electrophoresis, transferring them to a membrane, and hybridizing them with specific probes (probe A and probe B). The probes will bind to complementary sequences, allowing visualization of specific DNA fragments.
4. Step 4: Determine the possible genotypes of the children based on the parents' genotypes. Since the disorder is autosomal recessive, the parents are likely heterozygous (carriers) for the mutant allele. The children could inherit one of three genotypes: homozygous wild-type (WT/WT), heterozygous (WT/Mut), or homozygous mutant (Mut/Mut). Each genotype will produce a distinct band pattern on the gel.
5. Step 5: Draw the gel electrophoresis band patterns for each genotype. For homozygous wild-type (WT/WT), the band pattern will correspond to the fragment sizes of the wild-type allele. For heterozygous (WT/Mut), the band pattern will show fragments from both the wild-type and mutant alleles. For homozygous mutant (Mut/Mut), the band pattern will correspond to the fragment sizes of the mutant allele. Use the restriction maps and probe binding sites to determine the exact fragment sizes and their positions on the gel.

Key Concepts

Autosomal Recessive Inheritance

Autosomal recessive inheritance refers to a pattern where two copies of a mutated gene (one from each parent) are necessary for an individual to express a trait or disorder. In the case of alkaptonuria, both parents must carry at least one copy of the mutated HAO gene for their children to potentially inherit the disorder. This means that unaffected parents can still pass on the mutation to their offspring.

Restriction Enzymes and Restriction Mapping

Restriction enzymes, like BamHI, are proteins that cut DNA at specific sequences, allowing researchers to analyze genetic material. Restriction mapping involves determining the locations of these cut sites within a DNA molecule, which can help identify differences between alleles, such as the presence of mutations. In this scenario, the alteration of the BamHI site in the mutant allele is crucial for understanding how the mutation affects 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 fragments to a membrane for hybridization with labeled probes. This method is essential for analyzing the genotypes of the family members in the context of alkaptonuria, as it allows for the visualization of the presence or absence of specific alleles.