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Key Concepts 1. The construction of a genomic library 2. The difference between a cDNA library and a genomic library 3. The procedures involved in screening libraries by hybridisation 4. Amplification of DNA by PCR and gel electrophoresis 5. Applications of PCR technology
Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential B Biology
Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential B Biology
Plasmid clone
1. DNA is isolated from an organism and cut into thousands of pieces with restriction enzymes. 2. Plasmids or BACs are cut with the same restriction enzymes, and mixed with the genomic DNA fragments 3. DNA ligase is added and covalently links the two pieces of DNA together. plasmid or BAC DNA is used to 4. The recombinant p transform bacterial cells. 5. The collection of the thousands of clones of bacteria or BACs is a genomic library.
Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential B Biology
mRNA
Reverse transcriptase 3
Poly-A tail
A A A A A A3 T T T T T 5
5 3 DNA polymerase
5 3
3 5 cDNA
DNA hybridisation allows us to sort through the thousands of clones in a library and find a gene of interest. We can detect the gene of interest by its ability to basepair with a complementary sequence of a another DNA molecule. DNA probe single stranded labelled DNA fragments with the similar sequence as a gene of interest. The probe is tagged with a radioactive or a fluorescent label.
DNA probe
After separating the ds target DNA the ss probe will hydrogen bond to the complementary sequence.
Gene of interest
Nylon membrane
Gel electrophoresis
Separates macromolecules (DNA or proteins) on the basis of their rate of movement through a gel in an electrical field - molecular sieve sieve. The rate of movement depends on size, electrical charge, and other physical properties of the macromolecules. DNA separation depends mainly on size (length of f fragment) t) with ith longer l fragments f t migrating i ti less l along l the gel. Size markers, DNA of known size are used for calibration.
Lecture 2 BIOSCI 101 Essential B Biology Lecture 2 BIOSCI 101 Essential Biology
Gel electrophoresis
Lecture 2 BIOSCI 101 Essential Biology