Southern blotting workflow Which description best summarizes the core steps of a Southern blot used to detect specific DNA sequences?
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AElectrophoresis of DNA molecules and then blotting the separated DNA bands followed by incubation
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BElectrophoresis of DNA molecules and then blotting the separated RNA bands followed by incubation
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CElectrophoresis of DNA molecules followed by incubation without transfer
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DElectrophoresis of RNA molecules and then blotting the separated RNA bands followed by incubation
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EElectrophoresis of proteins followed by immunodetection
Answer
Correct Answer: Electrophoresis of DNA molecules and then blotting the separated DNA bands followed by incubation
Explanation
Introduction / Context:Southern blotting is a classic technique for detecting specific DNA fragments after restriction digestion and gel electrophoresis, using hybridization with a labeled probe.
Given Data / Assumptions:
- Target nucleic acid is DNA.
- Fragments are separated on an agarose gel.
- Transfer to a membrane enables stable probe hybridization.
Concept / Approach:The method proceeds: digest DNA, electrophorese, depurinate/denature, transfer (capillary or electroblot) to a membrane, fix DNA, hybridize with labeled probe, wash, and detect.
Step-by-Step Solution:Run DNA on agarose gel to resolve sizes.Denature and transfer DNA to a membrane (nylon/nitrocellulose).Hybridize with a complementary labeled DNA/RNA probe.Wash and visualize by autoradiography or chemiluminescence.
Verification / Alternative check:Controls with known positive and negative fragments confirm specificity and size.
Why Other Options Are Wrong:Options involving RNA describe Northern blotting; skipping transfer prevents durable detection; proteins correspond to Western blots.
Common Pitfalls:Poor transfer efficiency; nonstringent washes causing background; probe mishybridization.
Final Answer:Electrophoresis of DNA molecules and then blotting the separated DNA bands followed by incubation.