Q.11 The technique used to identify the specific DNA in bacterial colonies is-
I. In-situ hybridization
2. Dot blot
3. Colony hybridization
4. Western hybridization
Answer: Option 3 (Colony hybridization, ID: 68019147823).
Colony hybridization screens bacterial colonies grown on agar plates by replica plating onto nitrocellulose filters, lysing cells, hybridizing with labeled DNA probes specific to target sequences, and detecting via autoradiography.
Option Analysis
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1. In-situ hybridization: Detects nucleic acids within intact cells/tissues using probes; visualizes location but doesn’t screen bacterial colonies.
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2. Dot blot: Spots purified DNA/RNA directly on membrane for hybridization; tests extracts, not intact colonies.
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3. Colony hybridization: Transfers colonies to filter, lyses bacteria, denatures DNA in place, hybridizes with radioactive probe—identifies specific DNA-containing colonies.
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4. Western hybridization: Detects proteins via antibodies (Western blot); irrelevant for DNA identification.
Technique Comparison Table
| Technique | Target | Method | Screens Colonies? |
|---|---|---|---|
| In-situ hybridization | DNA/RNA in cells | Probe on tissue sections | No |
| Dot blot | Purified DNA/RNA | Direct spotting | No |
| Colony hybridization | Specific DNA sequences | Colony replica + probe | Yes |
| Western blot | Proteins | Antibody detection | No |
Colony hybridization technique bacterial colonies DNA screening is essential GATE Life Sciences knowledge for recombinant DNA technology questions. This method identifies colonies containing specific gene inserts using labeled probes.
Colony Hybridization Protocol (Grunstein-Hogness Method)
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Master plate: Grow transformed bacteria (e.g., E. coli with plasmids) on agar
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Replica plating: Press nitrocellulose filter onto colonies
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Lysis/denaturation: NaOH lyses cells, single-strands DNA binds filter
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Hybridization: Add ³²P-labeled probe complementary to target sequence
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Autoradiography: Dark spots = colonies with specific DNA
Why Colony Hybridization for Bacterial Screening?
| Purpose | Technique Choice | Application |
|---|---|---|
| Screen 10⁴ colonies/hour | Colony hybridization | Gene library screening |
| Tissue gene expression | In-situ hybridization | Cancer diagnostics |
| Quantify purified DNA | Dot blot | Southern blot confirmation |
| Protein detection | Western blot | Expression analysis |
GATE Life Sciences tip: “Specific DNA in bacterial colonies” = colony hybridization (developed 1975). Master this for molecular biology and microbiology PYQs—links perfectly to your membrane biology/biochemistry series.
