Figures A and B respectively represent the dideoxy sequencing gels obtained for partial sequences from 5′-ends of a bacterial gene and its mutant (with a point mutation)
What type of mutation this occurred in the gene ?
A. Nonsense
C. Frameshift
B. Missense
D. Transversion

Frameshift Mutation in Gene Sequencing

Gene mutations are alterations in the nucleotide sequence of DNA, which can have a significant impact on gene expression and protein function. One of the most disruptive types of mutations is a frameshift mutation, which occurs due to the insertion or deletion of nucleotides that are not in multiples of three. This type of mutation shifts the reading frame of the gene, leading to changes in the downstream amino acid sequence and often resulting in nonfunctional proteins.

In this case, Figures A and B represent the dideoxy sequencing gels obtained for partial sequences from the 5′-ends of a bacterial gene and its mutant with a point mutation. The correct interpretation of the sequencing data indicates that a frameshift mutation has occurred. Let’s explore why frameshift mutation is the correct answer and how it affects gene function.

Correct Answer:

The correct answer is C. Frameshift.

What is a Frameshift Mutation?

A frameshift mutation occurs when there is:

• Insertion or deletion of one or more nucleotides in a sequence.
• If the number of inserted or deleted nucleotides is not divisible by three, the reading frame of the codons is altered.
• This results in the translation of incorrect amino acids, leading to a dysfunctional protein or a premature stop codon.

How Frameshift Mutation Is Detected Using Dideoxy Sequencing

1. Principle of Dideoxy Sequencing (Sanger Sequencing):

• Sanger sequencing uses dideoxynucleotides (ddNTPs) that lack a 3′-OH group.
• When a ddNTP is incorporated during DNA synthesis, it terminates chain elongation.
• The resulting fragments are separated by size using gel electrophoresis, and the sequence is read from the gel.

2. Analysis of Wild-Type Sequence (Figure A):

• In the wild-type gel, the sequence will show a normal and consistent pattern.
• Each band corresponds to the position of a nucleotide incorporated into the growing strand.

3. Analysis of Mutant Sequence (Figure B):

• In the mutant gel, the pattern will shift after the point mutation.
• A missing or extra band indicates an insertion or deletion of a nucleotide.
• The shift in the pattern confirms that the mutation is altering the reading frame.

4. Identification of Frameshift:

• A shift in the sequence after the mutation site confirms that the codon reading frame is disrupted.
• This is a hallmark of a frameshift mutation.

Why Other Options Are Incorrect

How Frameshift Mutation Affects Protein Synthesis

1. Altered Reading Frame:

   • The mutation changes the grouping of nucleotides into codons.
   • Incorrect codons lead to altered amino acid sequences.

2. Premature Stop Codon:

   • The shift may create a stop codon prematurely, truncating the protein.
   • This results in a nonfunctional or incomplete protein.

3. Loss of Function:

   • The protein may lose its biological activity due to incorrect folding.
   • In severe cases, the mutation can cause genetic disorders or loss of enzyme function.

Example of Frameshift Mutation

Wild-Type Sequence:

5’-AUG CCG GAA UCC GAU-3’

• Encodes: Met-Pro-Glu-Ser-Asp

After Frameshift Mutation:

5’-AUG CCG GAU CCG AU-3’

• Encodes: Met-Pro-Asp-Pro
• The reading frame is shifted, altering the downstream amino acid sequence.

Consequences of Frameshift Mutations

 Loss of protein function
 Protein misfolding
Creation of a nonfunctional protein
 Potential to cause genetic diseases

Applications of Dideoxy Sequencing in Mutation Detection

1. Identifying Genetic Disorders:

• Dideoxy sequencing helps identify frameshift mutations linked to diseases like cystic fibrosis and Duchenne muscular dystrophy.

2. Cancer Research:

• Frameshift mutations in tumor suppressor genes (like p53) can lead to cancer progression.

3. Drug Resistance Studies:

• Frameshift mutations in bacterial genes can confer antibiotic resistance.

Challenges in Detecting Frameshift Mutations

1. Short Reads:

• Dideoxy sequencing is limited to short sequences (100–1000 bp).

2. Mixed Populations:

• Mixed template sequences can make interpretation difficult.

3. Background Noise:

• Misincorporation of nucleotides can create artifacts in the sequencing gel.

Why Frameshift Mutation Is Disruptive

• The entire downstream reading frame is altered.
• Protein structure and function are drastically changed.
• Loss of function or gain of toxic function can lead to disease.

Conclusion

Frameshift mutations occur when nucleotide insertions or deletions change the reading frame of a gene. Dideoxy sequencing allows the detection of these mutations by revealing shifts in the sequence pattern. In the case of Figures A and B, the altered gel pattern confirms the presence of a frameshift mutation. This type of mutation can have severe consequences, including loss of protein function and the development of genetic diseases.