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#colony hybridization

3 public questions tagged with this topic.

Transcriptome analysis

A hybridization-based method that uses mRNA extracted from colonies on a membrane is:

Colony hybridization involves transferring bacterial colonies or phage plaques from agar plates onto nitrocellulose or nylon membranes, lysing them to release nucleic acids, and immobilizing DNA or RNA onto membrane surface. Labeled probes specific to target mRNA or genes are hybridized to filter, allowing screening of many clones simultaneously. This method was pivotal for library screening before high-throughput sequencing. FISH performs in situ hybridization on chromosomes, northern blot involves gel electrophoresis, and CAGE captures 5′ caps, none employing colony lysis on membranes for mR

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Cloning Process

In colony hybridization, what detects hybridization?

After transfer and fixation of colony DNA to membrane, hybridization with radiolabeled probe specific for insert is carried out. To visualize successful annealing, membranes are exposed to X-ray film sensitive to beta radiation emitted by isotopes such as 32P incorporated into probe backbone. Regions where probe hybridized produce dark spots on film due to autoradiographic emission, aligning with original colony positions. This X-ray autoradiography provides high sensitivity and spatial resolution. Detection does not rely on ultraviolet fluorescence, colorimetric X-gal cleavage, or direct fluo

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details
Gene Cloning Process

Which method uses radioactive probes to identify recombinant colonies?

Colony hybridization, developed by Grunstein and Hogness, identifies recombinant clones harboring specific DNA sequences without phenotypic screening. Colonies are replica-transferred to nitrocellulose or nylon membrane, lysed with alkali, and DNA denatured and fixed. The membrane is then incubated with a 32P or 35S labeled nucleic acid probe complementary to desired insert. After stringent washing to remove non-specific binding, positive colonies are detected by autoradiography where hybridization signal exposes X-ray film. Blue-white screening and direct antibiotic selection do not employ ra

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details