Q.54 Identify the correct sequence of steps involved in detection of specific DNA fragments
from a mixture of DNA:
- DNA is separated by electrophoresis and visualized by staining.
- Filter is placed in sealed bag with solution containing radioactive probe.
- DNA samples are cut by Restriction Endonucleases and loaded onto gel.
- Filter is washed to remove excess probe and placed on X-ray film to produce images of DNA bands.
- DNA binding filter paper towels and weights are placed on gel.
Due to capillary action DNA fragments bind to the membrane.
Choose the correct answer from the options given below:
- C – A – E – B – D
- A – B – C – D – E
- E – D – C – B – A
- A – B – D – C – E
C – A – E – F – G – B – D represents the complete correct sequence for Southern blotting, though the options provided follow this core order with C – A – E – B – D as the closest match.
Question Breakdown
This question tests the Southern blotting technique steps for detecting specific DNA fragments from genomic DNA mixtures—a cornerstone molecular biology method developed by Edwin Southern in 1975.
Correct Sequence Explanation (Mapping Statements)
C → A → E → F → G → B → D (Note: F=”Due to capillary action…” and G=”DNA binding filter…” appear combined in options):
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C: DNA samples cut by restriction endonucleases generates specific fragments (EcoRI, HindIII).
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A: Electrophoresis separates fragments by size; ethidium bromide/UV visualizes all DNA (pre-transfer check).
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E: DNA binding filter paper towels and weights assemble capillary transfer stack (gel → membrane → paper towels).
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F+G: Capillary action transfers single-stranded DNA from gel to nylon/nitrocellulose membrane.
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B: Radioactive probe hybridization in sealed bag detects specific sequences.
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D: Washing removes unbound probe; autoradiography reveals target bands.
Option C – A – E – B – D correctly captures the core workflow, treating F/G as implicit transfer steps.
Why Other Options Are Wrong
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A – B – C – D – E: Electrophoresis (A) before digestion (C); DNA must be fragmented first.
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E – D – C – B – A: Transfer setup (E) before digestion (C); sequence starts with sample prep.
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A – B – D – C – E: Hybridization (B) before digestion (C); impossible reverse order.
The Southern blot steps sequence enables specific DNA fragment detection from complex mixtures, revolutionizing gene mapping and diagnostics. This guide details the correct sequence of steps involved in detection of specific DNA fragments—C (restriction digestion) → A (electrophoresis) → E (transfer setup) → B (probe hybridization) → D (detection)—essential for CSIR-NET, GATE Life Sciences preparation.
Southern Blotting Principle
Restriction fragments → agarose gel separation → membrane transfer → hybridization → visualization. Detects single-copy genes amid 3×10⁹ bp genomes.
Step-by-Step Protocol (C-A-E-B-D)
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C. Restriction digestion: EcoRI/HindIII cut DNA at specific sequences (4-6 bp recognition).
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A. Gel electrophoresis: 0.8% agarose, TAE buffer, 5 V/cm separates 0.1-10 kb fragments.
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E. Capillary transfer stack: Gel → nylon membrane → 3MM paper → paper towels + 500g weight (16-24 hrs).
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B. Hybridization: ³²P/Chemiluminescent probe (10⁶ cpm/mL), 65°C overnight rotation.
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D. Stringent washing + autoradiography: 0.1× SSC/0.1% SDS, 65°C; X-ray film exposure 24-72 hrs.
| Step | Purpose | Key Reagents | Time |
| C | Generate specific fragments | Restriction enzymes | Overnight |
| A | Size separation | Agarose, EtBr | 4-6 hrs |
| E+F+G | Transfer to membrane | 20× SSC, nylon | Overnight |
| B | Specific detection | ³²P-labeled probe | Overnight |
| D | Visualization | X-ray film | 1-3 days |
This DNA fragment detection sequence remains gold standard for RFLP analysis, paternity testing, and forensic DNA profiling.
