A Molecular Toolkit AP Biology Fall 2010

The Scissors: Restriction Enzymes  Bacteria possess restriction enzymes whose usual function is to cut apart foreign DNA molecules

The Scissors: Restriction Enzymes  Each enzyme cuts only at sites that possess a specific base sequence The wide variety of restriction enzymes and their specificity makes it possible to study the genome of a particular species Many times the “stick ends” that result from the cut can be used to pair up with another DNA fragment cut by the same enzyme

The Scissors: Restriction Enzymes  DNA fragments produced by restriction enzymes are treated with DNA ligase to splice the DNA fragments (from same or different species) together to form a recombinant DNA molecule

Cloning Vectors  Plasmids: circular DNA molecules in bacteria that carry only a few genes and can replicate independently of the single “main” chromosome

Cloning Vectors  Cloning vectors: modified plasmids that are capable of accepting, replicating, and delivering DNA to another host cell

Cloning Vectors  When the plasmid is replicated, any foreign DNA that may have become incorporated into it is also replicated Produces a DNA clone

cDNA Cloning  Even after a desired gene has been isolated and amplified, it may not be translated into functional protein by the bacteria because introns (noncoding regions) are still present

cDNA Cloning  Researchers minimize this problem by using cDNA, which is made from “mature” mRNA transcripts The cDNA is made from mRNA by reverse transcriptase The cDNA can be inserted into a plasmid for amplification