Colony hybridization for DNA sequence identification
The technique used to identify specific DNA sequence in bacterial colonies is
(a) Colony hybridization
(b) In situ hybridization
(c) Dot blot technique
(d) Western blotting
The Technique Used to Identify Specific DNA Sequence in Bacterial Colonies
The correct technique used to identify a specific DNA sequence in bacterial colonies is (a) Colony Hybridization.
What is Colony Hybridization?
Colony hybridization is a powerful molecular biology technique that enables researchers to screen bacterial colonies for the presence of a specific DNA sequence. This method is widely used in recombinant DNA technology, where scientists need to determine whether a bacterial colony contains the desired gene insert.
By using labeled DNA probes that bind to complementary DNA sequences, colony hybridization for DNA sequence identification allows efficient screening of bacterial colonies carrying the target gene.
Steps of Colony Hybridization for DNA Sequence Detection
1. Bacterial Colony Growth and Plating
- Bacteria containing recombinant plasmids are grown on an agar plate with selective antibiotics.
- After incubation, bacterial colonies form, each derived from a single transformed bacterial cell.
2. Transfer of Bacterial Colonies onto a Membrane
- A nitrocellulose or nylon membrane is gently placed over the bacterial colonies to create a replica of the plate.
- The membrane is treated to lyse bacterial cells, exposing and denaturing the DNA into single strands.
3. Hybridization with a Labeled Probe
- A radioactive, fluorescent, or chemiluminescent-labeled DNA probe complementary to the target DNA sequence is introduced.
- The probe binds specifically to complementary DNA sequences in colonies that contain the gene of interest.
4. Detection of Hybridized Colonies
- Excess probes are washed away, leaving only those bound to complementary sequences.
- The membrane is analyzed using autoradiography (for radioactive probes) or fluorescent detection (for fluorescent probes).
- Bacterial colonies containing the desired DNA sequence appear as dark spots on an X-ray film or under UV light.
Applications of Colony Hybridization for DNA Sequence Identification
✔ Screening bacterial colonies for recombinant DNA
✔ Identifying specific genes in a gene library
✔ Verifying gene insertion in bacterial clones
✔ Detecting disease-related genes in medical and forensic applications
Why Not Other Techniques?
1. In Situ Hybridization (ISH)
- Used to detect nucleic acid sequences within tissues and cells, not bacterial colonies.
2. Dot Blot Technique
- Useful for detecting DNA or RNA sequences but does not allow identification in individual bacterial colonies.
3. Western Blotting
- Used to detect proteins using antibodies, not DNA sequences.
