Molecular Bio Exam 2

DNA Cloning with restriction enzymes

DNA cloning is used to make large copy numbers of a specific segment of DNA; done by putting DNA of interest into a self-replicating DNA molecule, and then “growing this DNA in cells”; commonly done by putting DNA in a plasmid (vector)

Restriction enzymes

Also called restriction endonucleases; cut DNA; recognize and bind to specific DNA sequences (called restriction site); Type II most important for DNA cloning

Types of restriction enzymes

• Type I - randomly cleaves DNA around 1000bp away from restriction site

• Type II - cleave within or a short specific distance from restriction site

• Type III - cleave 24-26bp away from restriction site

• Type IV - recognize and cleave modified DNA

Insert DNA

Two common types:

• Using natural occurring restriction sites - cutting chromosomal DNA at the naturally occurring restriction sites to clone a specific region of DNA

• Adding restriction sites via PCR - using PCR to add restriction enzyme sites and amplify a specific piece of DNA

Plasmid features

• Selection markers - Allows scientists to selectively grow cells with the plasmid

• Origin of replication - Allows the plasmid to be replicated so it can be propagated

• Unique Restriction Enzyme Sites - Sites that allow the plasmid to be cut to allow inserting foreign DNA