Bio102: Introduction to Cell Biology and Genetics
Recombinant DNA
Key Terms:
gene cloning
restriction enzyme
sticky ends
plasmid
ligation
ligase
plasmid library
recombinant DNA
PCR
transformation
primer
Taq DNA polymerase
cDNA
reverse transcriptase
expression vector
Key Questions:
 What is a clone?
 What important features are needed on a cloning plasmid? on an expression plasmid?
 What is meant by a ‘plasmid library’?
 In PCR, no helicase is needed. Why not?
 How is the target gene to be amplified in PCR selected?
Lecture Outline:
Steps to Clone a Gene:
Isolating the DNA of interest
isolate total DNA from human cell. cut DNA into manageable fragments with a restriction enzyme
generates sticky ends
Ligate to Plasmid Vector. Sticky ends stick to one another
fix the gap with DNA ligase
now have a collection of many different fragments in the plasmid; a “library”
Transform into a host, usually E coli
plasmid has an antibiotic resistance gene. only cells getting plasmid live and form colonies
Identifying the Correct Clone
depends on the gene you’re targeting.
if it’s an enzyme, may be able to do enzyme assay on each strain to find one with your gene
Polymerase Chain Reaction (PCR)
essentially DNA replication in a test tube
strands separated with heat. cool to allow short DNA primers to bind. Polymerase extends primers
repeat many times for exponential increase in copy number
now clone this DNA into a plasmid, like before
Expressing eukaryotic proteins in prokaryotes
remove introns from the gene
isolate mRNA from the cytoplasm so the eukaryotic cell has already removed introns
convert mRNA to DNA with the enzyme reverse transcriptase
this is a copy DNA (cDNA) which will lack intron sequences
provide prokaryotic promoter and Shine-Dalgarno sequence on expression vector