Chapter 12.1-12.2: PCR, Cloning, and Plasmids

PCR synthesizes

large quantities of a DNA fragment

application of PCR

gene cloning

taq polymerase

enzyme that builds new DNA strands during

PCR

where does taq polymerase come from

heat-loving bacterium (thermostable)

denaturing

targeting DNA with heat

annealing

primers bind to target DNA

extension

copies of target DNA are synthesized

molecular cloning

isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated

first step of molecular cloning

isolation and fragmentation of source DNA

second step of molecular cloning

insertion of DNA fragment into cloning vector

third step of molecular cloning

introduction of cloned DNA into host organisms

restriction endonuclease

cut DNA at specific sequences and create ends that allow insertion into plasmids

DNA ligase

forms recombinant molecules (combines ends)

plasmids as cloning vectors are easy

to purify small size

PCR purpose

to amplify DNA

steps of PCR

denature - anneal - extend

PCR cloning

DNA inserted into vector, put into host cell, and produces lots of DNA