Download 22. Recombinant DNA Technology

Recombinant DNA Technology
Restriction endonucleases - Blunt ends and Sticky ends
Restriction endonuclease and DNA ligase yield Recombinant DNA
DNA cloning
Polylinker – multiple restriction sites
Selection of clones
Bacterial Artificial Chromosomes (BAC)
Transformation:
1. Heat shock: CaCl2 at 0oC then heat to
37-42oC
2. Electroporation – apply high voltage
BAC – 5,000 to 400,000 bp insert
Yeast Artificial Chromosomes (YAC)
up to 150,000 bp insert
Studying genes – cDNA library
Polymerase Chain Reaction (PCR)
Cloning of PCR products
Hybridization allows the deletion of specific sequences
DNA fingerprinting – RFLP (restriction fragment length
polymorphism)
DNA microarrays
Any known DNA sequence from any source, can be
used in microarray.
Green spots – mRNA more abundant in single-cell stage
Red spots – mRNA more abundant at later stages of
development
Cloned genes can be expressed – Expression vector
Cloned genes can be altered
1.
Site-directed
mutagenesis
2.
Oligonucleotidedirected mutagenesis
Transgenic – cloning in mice for human growth hormone