You are working in a lab where you are studying a disease that is known to be caused by a single nucleotide change, although the effect this change ultimately has on the protein's structure/function is unknown. You have DNA samples from multiple patients that you suspect of having this disease. What is the most efficient way to test the samples for the relevant mutation?单项选择题
A
Western blotting
B
DNA sequencing
C
Karyotyping
D
Amniocentesis

Question

You are working in a lab where you are studying a disease that is known to be caused by a single nucleotide change, although the effect this change ultimately has on the protein's structure/function is unknown. You have DNA samples from multiple patients that you suspect of having this disease. What is the most efficient way to test the samples for the relevant mutation?单项选择题

A

Western blotting

B

DNA sequencing

C

Karyotyping

D

Amniocentesis

BlackTom AI · Accepted Answer

DNA sequencing reads the nucleotide sequence directly, so it will reveal any single-nucleotide change (point mutation) in the patient's DNA.

1) Relevant knowledge points and concepts
- A single-nucleotide change is a point mutation; detection requires methods that can resolve single-base differences.
- DNA sequencing (Sanger or targeted NGS) determines the exact base at each position in a defined region and therefore directly detects point mutations.
- Western blotting measures protein presence/size, karyotyping detects large chromosomal rearrangements or aneuploidy, and amniocentesis is a sampling procedure for prenatal diagnosis—not a detection technology for point mutations in general patient samples.

2) Step-by-step reasoning process
- You have DNA samples and suspect a specific disease-causing nucleotide change. The most direct approach is to sequence the genomic region containing the candidate nucleotide.
- Extract genomic DNA from each sample.
- Design PCR primers flanking the suspected mutation site and amplify the target region.
- Perform Sanger sequencing (efficient and cost-effective for a single locus across multiple samples) or targeted NGS if higher throughput is needed.
- Analyze chromatograms/sequence reads at the nucleotide position to determine whether each sample carries the reference or altered base.

3) Intermediate steps (for a typical workflow)
- DNA extraction → quantify DNA.
- PCR amplification of ~200–800 bp surrounding mutation.
- Clean PCR product → Sanger sequencing reaction → capillary electrophoresis → read chromatogram.
- Inspect base calls: heterozygous change shows overlapping peaks; homozygous change shows a single altered peak.

4) Why the correct answer is correct
Sequencing directly interrogates the DNA at single-base resolution, so it will unambiguously detect the specific single-nucleotide change whether it alters protein sequence, expression, or has no known protein effect.

5) Why the other options are incorrect
- Western blotting: measures protein size/abundance; can miss point mutations that do not change protein size, stability, or expression.
- Karyotyping: visualizes whole chromosomes and detects large structural changes (>5–10 Mb), not single-nucleotide variants.
- Amniocentesis: a prenatal sampling procedure, not a detection method; also invasive and irrelevant for routine testing of patient DNA samples.

6) Conclusion
DNA sequencing is the most efficient and appropriate method to test these samples for a single-nucleotide mutation. This matches the provided correct answer: DNA sequencing.

类似问题

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