Amino Acid Variants in Sanger Sequencing of Human Hemoglobin

Representations of sequencing gels obtained from Sanger’s dideoxy chain termination method for variants A and B of the a chain of human hemoglobin are shown here. What is the nature of the amino acid’?

A. Residue 2, L R,CTG -i CGG
B. Residue 5, A -i P,GCC -Y CCC
C. Residue 6, D G,GAC GGC
D. Residue 6, Q P,CAG CCG

Understanding Amino Acid Variants in Sanger Sequencing of Human Hemoglobin

Sanger’s dideoxy chain termination method is a widely used technique for sequencing DNA and identifying mutations or variations within genes. It allows precise determination of nucleotide sequences, which helps in detecting single nucleotide polymorphisms (SNPs) and amino acid substitutions in proteins.

In the case of human hemoglobin, mutations can lead to significant changes in protein structure and function, often resulting in genetic disorders like sickle cell anemia or thalassemia. The sequencing gels obtained from Sanger sequencing provide valuable insights into these mutations.

Correct Answer:

👉 The correct answer is D. Residue 6, Q P, CAG → CCG

This represents a substitution of glutamine (Q) with proline (P) at residue 6 in the hemoglobin chain due to a single nucleotide mutation from CAG to CCG.
This type of mutation can lead to altered hemoglobin function, affecting oxygen transport efficiency and protein stability.

Sanger Sequencing and Its Importance

Sanger sequencing, developed by Frederick Sanger in 1977, is a chain termination-based method used to determine the nucleotide sequence of DNA. It relies on the use of:
Dideoxynucleotides (ddNTPs): Cause chain termination during DNA synthesis.
Fluorescent or radioactive labeling: Allows detection of terminated fragments on a gel or capillary electrophoresis.
Gel electrophoresis: Separates fragments based on size to determine the DNA sequence.

How Sanger Sequencing Works

Template DNA is denatured into single strands.
Primers bind to the template strand to initiate synthesis.
A mix of dNTPs (normal nucleotides) and ddNTPs (terminating nucleotides) is added.
DNA polymerase incorporates dNTPs until a ddNTP is inserted, terminating the chain.
The resulting fragments are separated by size using gel electrophoresis.
The DNA sequence is determined by reading the gel pattern from smallest to largest fragments.

Nature of the Mutation in Hemoglobin

Residue 6, Q P, CAG → CCG

CAG codes for glutamine (Q), while CCG codes for proline (P).
This is a missense mutation where a single nucleotide change causes a change in the amino acid sequence of hemoglobin.
Substitution of glutamine with proline may lead to structural instability and altered hemoglobin function.

Explanation of Other Options

A. Residue 2, L R, CTG → CGG

CTG codes for leucine (L), and CGG codes for arginine (R).
This is a missense mutation, but it’s not related to the mutation observed in the gel.

B. Residue 5, A → P, GCC → CCC

GCC codes for alanine (A), and CCC codes for proline (P).
Though this reflects a substitution, it does not correspond to the observed gel pattern.

C. Residue 6, D G, GAC → GGC

GAC codes for aspartic acid (D), and GGC codes for glycine (G).
This mutation is different from the one observed at residue 6 in hemoglobin.

Consequences of Q to P Substitution in Hemoglobin

Structural Changes:
- Proline introduces a kink in the protein structure due to its cyclic side chain.
- This may affect the stability and folding of the hemoglobin molecule.
Functional Impact:
- Altered hemoglobin structure may reduce oxygen-binding efficiency.
- Structural rigidity could impair the cooperative binding mechanism.
Pathological Impact:
- Missense mutations in hemoglobin are associated with disorders like:
  - Sickle Cell Anemia (mutation in β-globin)
  - Thalassemia (reduced or absent globin chain production)

Why Proline Substitution is Critical

Proline has a unique cyclic structure that limits flexibility.
Insertion of proline in place of glutamine can lead to structural distortions.
This change may interfere with hemoglobin’s ability to bind oxygen properly.

Techniques to Confirm Mutation

PCR (Polymerase Chain Reaction): Amplifies the mutated DNA for further analysis.
Mass Spectrometry: Confirms the altered amino acid sequence.
Western Blotting: Detects changes in hemoglobin protein expression.

Clinical Relevance of Hemoglobin Mutations

Sickle Cell Anemia:
- Caused by a missense mutation in the β-globin gene (Glu → Val).
- Leads to hemoglobin polymerization and distorted red blood cells.
Thalassemia:
- Caused by mutations in the α or β-globin genes.
- Results in reduced or absent hemoglobin production.
Hemoglobinopathies:
- Structural or functional changes in hemoglobin.
- Can lead to anemia and other blood disorders.

Applications of Sanger Sequencing in Medicine

Genetic Testing: Identification of inherited disorders.
Pharmacogenomics: Tailoring drug therapy based on genetic profile.
Cancer Research: Identifying driver mutations and therapeutic targets.
Prenatal Diagnosis: Detecting genetic abnormalities in the fetus.

Conclusion

Sanger sequencing remains a powerful tool for detecting mutations in genes, including those involved in hemoglobinopathies. The substitution of glutamine (Q) with proline (P) at residue 6 in human hemoglobin (CAG → CCG) represents a missense mutation that may affect hemoglobin’s structural integrity and function. Understanding these genetic variations is crucial for diagnosing and managing hemoglobin-related disorders.