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. Oligonucleotide-directed mutagenesis

Transgenic – cloning in mice for human growth hormone