DNA cloning

•  Act of making many identical copies

•  Cell-based DNA cloning

•  Cell-free DNA cloning (PCR)

Basic steps in DNA cloning
•  Identify and isolate specific DNA

•  Insert DNA into vector

•  Clone in dividing cells

•  Isolate cloned DNA

Basic steps in DNA cloning
•  Identify and isolate specific DNA

•  Insert DNA into vector

•  Clone in dividing cells

•  Isolate cloned DNA

 DNA ligase
•  Join together any two DNA fragments

•  DNA can be from different organisms

Basic steps in DNA cloning
•  Identify and isolate specific DNA

•  Insert DNA into vector

•  Clone in dividing cells

•  Isolate cloned DNA

 Vector
•  Capable of accepting a foreign DNA
fragment
•  Capable of replicating itself
•  Most common are plasmids
•  Origin of replication
•  Antibiotic resistance genes
Insertion of DNA into plasmid

cleave with RE
insert DNA with
DNA ligase
Basic steps in DNA cloning
•  Identify and isolate specific DNA

•  Insert DNA into vector

•  Clone in dividing cells

•  Isolate cloned DNA

Clone in dividing cells
Basic steps in DNA cloning
•  Identify and isolate specific DNA

•  Insert DNA into vector

•  Clone in dividing cells

•  Isolate cloned DNA

 Isolate cloned DNA
•  Detect cells containing recombinant DNA

•  Antibiotic resistance

•  “Blue-white” screening
Cloning of human genes
•  Factor VIII - hemophilia

•  Insulin - diabetes
Genomic DNA library
•  Coding sequences

•  Intronic sequences

•  Gene regulatory regions

•  Repetitive DNA sequences

 cDNA library
•  Coding sequence only

•  Tissue specific gene sequences

Polymerase Chain Reaction
(PCR)
•  Developed in the mid 1980s
•  Revolutionized molecular biology
•  DNA fragments can be amplified in large
amounts
•  In vitro technique
•  Kary Mullis - Nobel prize (1993)
What do we need for PCR?
•  Sequence information
•  Oligonucleotide primers
•  DNA
•  Nucleotides
•  Heat-stable DNA polymerase
• Taq (Thermus aquaticus)
 PCR technique
ds DNA
Step 1 Denature

Step 2 Anneal

Step 3 Extend
PCR - Advantages
•  Speed

•  Ease of use

•  Sensitive

•  Robust
PCR - Disadvantages
•  Need sequence information

•  Limited amplification size

•  Limited amounts of product

•  Infidelity of DNA replication

PCR - Applications
•  DNA cloning
•  Detection of disease mutations
•  DNA sequencing
•  In-vitro mutagenesis
•  Gene identification
•  Gene expression studies
•  Forensic medicine
 Application of DNA
technology
•  Study the function of genes and proteins

•  Construct novel DNA molecules

•  Produce large amounts of protein -

insulin, factor VIII, growth hormone

•  Produce RNA in vitro

 Find a gene

What does it do?

 Identify the function
of a gene
•  Create a mutant

•  Site-directed mutagenesis

•  Re-introduce mutant into organism

•  Animal models
 Normal gene

Replacement Addition

Knockout
•  Gene replacement - info about the
mutant gene

•  Gene knockout - info about the normal

function of the gene

•  Gene addition - info about mutant

effects over normal gene
•  Identify a gene
•  Clone it
•  Mutate it
•  Create animal models
•  Find function
•  Understand biology
•  Develop drugs