PCR
Polymerase Chain Reaction - Number of DNA strands doubles
3 Steps of PCR
- Denature
- Annealing
- Synthesis
Denature
High heat separates DNA strands by breaking h-bonds
Annealing
low heat, add specific primers, they attach to the DNA strands
Synthesis
Taq polymerase and nucleotides added, medium heat. Polymerase complimentary base pairs and add 3’ end of the primer.
rtPCR
reverse transcription
2 Steps of rtPCR
- Primer attaches to a mRNA strand
2. Reverse transcriptase enzyme and DNA nucleotides added-complimentary DNA strand
Uses of rtPCR
- create cDNA libraries
- Create DNA to then make more of that protein
- rtPCR is used in the COVID test (SARS-CoV-2 is an mRNA virus)
Gel Electrophoresis will separate DNA fragments by
size
Gene cloning Vector
- ORI
- Marker Gene
- Multiple Cloning sites with restriction sites
Technique for DNA extraction
- Use of detergents to break down lipid membrane
- Protease enzymes, ribonuclease enzymes, break down proteins and RNA
- Alcohol to precipitate from the solution
Restriction Enzyme
cut DNA in specific spots
Technique for Gel electrophoresis
- Cut DNA
- Place DNA + loading dye sample into gel wall
- Hook gel up to electrical current
- Run until loading dye is about 3/4 through gel, then visualize with UV light.
Recombinant DNA
A piece of DNA with foreign DNA added to it
Transformation
When bacteria uptakes new DNA
Reproductive Cloning
Cloning of a full organism
Uses of DNA Sequencing
- Study genes and proteins
- Study evolution
- Study Communities
- Study viruses
- Medical Applications- testing/ diagnosis, forensics, paternity
