Restriction Endonucleases
Enzymes that break phosphodiester bonds within DNA molecules
Type I: Specific recognition site with nonspecific cleavage site a distance away
Type II: Specific recognition site with internal site cleavage
Type III: Specific recognition site with nearby nonspecific cleavage site
Probes
- Complement of the sequence for DNA/RNA
- DNA probes are more stable and preferred
- Target sequence must be single stranded
- Increasing length increases specificity and decreases efficiency
- Visualized through radioactive exposure on a film (P35 or C14) or biotinylated with a fluorescent dye
Blotting
Makes a stable copy of a gel transferring nucleic acid or protein to a nitrocellulose membrane
Southern: Used to separate DNA, usually based on length
Northern: Used to separate RNA, usually based on length
Western: Used to separate proteins based on various attributes and visualized using antibodies
A DNA molecule capable of replication in a host and into which a gene or piece of DNA can be specifically inserted at known positions
Cloning vector
Cloning Vector Steps
- Cut plasmid and target DNA with a restriction endonuclease that leaves sticky ends
- Mix plasmid and target DNA to allow sticky ends to connect
- Complete with ligase
Plasmid Cloning Vector Requirements
- Ori sequence
- Dominant selectable marker (e.g. antibiotic resistance)
- Unique restriction site inside a reporter gene
- A reporter gene (LacZ: blue when functional and exposed to X-Gal)
Lambda Cloning Vector
Engineered phage with only lytic capabilities that can insert DNA into bacteria
Cosmid Cloning Vectors
Engineered to combine phage and plasmid features
- Ori sequence
- Dominant selectable marker
- Unique restriction sites
BAC
Bacterial Artificial Chromosome: Remove all “production” DNA so that new DNA can be inserted and all nutrients can be added to media instead of produced
YAC
Yeast Artificial Chromosome: Used for cloning long genes
- Centromere
- Two telomeres
- Origin of replication
- Restriction sites to insert DNA
PCR
Polymerase Chain Reaction
- DNA is denatured (melted) at 95*C
- Primers anneal at 55*C
- Taq polymerase elongates strand at 70*C
VNTR
Variable Number of Tandem Repeats
- Repeated sequences in the genome that vary between people
- Can match tissue by visualized exact same repeat numbers
- Can match inheritance by having one band from each parent
