Lecture 204 of 373: CDNA Libraries: Hybridization Probing and Labelling with Radioactive Marker (31 mins) | CUET (Common University Entrance Test) PG Zoology (SCQP28) | Complete Video Course 373 Lectures [222 hrs : 42 mins]
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cDNA libraries
mRNA can be converted into DNA by complementary DNA (cDNA) synthesis.
The key to this method is the enzyme reverse transcriptase, which synthesizes a DNA polynucleotide complementary to an existing RNA strand.
Once the cDNA strand has been synthesized the RNA member of the hybrid molecule can be partially degraded by treating with ribonuclease (RNase) HI.
The remaining RNA fragments then serve as primers for DNA polymerase I, which synthesizes the second cDNA strand, resulting in a double-stranded DNA fragment that can be ligated into a vector and cloned.
Hybridization probing
Nucleic acid hybridization can be used to identify a particular recombinant clone if a DNA or RNA probe, complementary to the desired gene, is available.
Hybridization probing can be used to identify recombinant DNA molecules contained in either bacterial colonies or bacteriophage plaques.
Labelling with a radioactive marker
Nick translation
End filling
Random priming
Non-radioactive labelling
Labelling via biotin and avidin fluorescent tag
Labelling with HRP luminol assay
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