DNA Cloning

Cloning

• a line of genetically identical cells or individuals derived from a single ancestor

• produces many copies of a piece of DNA

• uses a little fraction as gene of interest

• cultivates a large amount for studying functions

Functions

• Biological• determine the DNA se

quence• ultimate information• regulation• Pharmaceutical• manufacture medicine

s• Clinical

How it Works

• Combine gene of interest and bacterial plasmid

• Recombinant DNA• DNA from two or mor

e different sources that have been joined together to form a single molecule

• Amplification and identification

Zoom In!!!

• Insertion, the most critical step

• made possible by restriction endonucleases(aka. restriction enzymes)

• recognize specific DNA sequences called restriction sites

• cut the DNA at specific locations

• Restriction fragments

Restriction Enzyme• defend against viral attack by

breaking down the DNA molecules of infecting viruses

• cleave the sugar-phosphate backbones of DNA to produce sticky ends

• short single-stranded regions• form hydrogen bonds with co

mplementary sticky ends on any other DNA molecules cut with the same enzyme

• kind of like glue when you fix something

Cloning Vectors

• DNA molecules into which a DNA fragment can be inserted to form a recombinant DNA molecule for the purpose of cloning

• plasmid cloning vectors

• contains 2 unique genes: ampR gene and lacZ+ gene

The Two Genes

• ampR gene• encodes an enzyme t

hat breaks down the antibiotic ampicillin

• lacZ+ gene• encodes ß-galactosid

ase (hydrolyzes the sugar lactorse and synthetic substrates)