4. In Vitro cloning Flashcards
list the features of in vitro cloning
- outside an organism
- uses PCR
- makes millions of fragments in a shorter time frame
- relies on sequencing to see which genes have been amplified
define PCR
the process of using primers to amplify a specific strand of DNA, to complete the artificial replication of a short sequence of DNA
define amplification
to make multiple copies of a specific DNA
in order to complete a PCR, what must the DNA be placed in
a buffer solution containing:
1. DNA nucleotides
2. Taq DNA
3. primers complimentary to the DNA
list the stages needed to heat and seperate DNA strands
- Denaturing
- Annealing
- Elongation
what is the rough temperature for Denaturing
95 degrees
what is the ideal temperature for annealing
55-60 degrees
what is the ideal temperature for elongation
72 degrees (optimum temperature for Taq polymerase)
outline denaturation
- mixture is heated
- hydrolyses the hydrogen bonds between the nucleotides, forming single strands of DNA
- separating the double strand of DNA
outline annealing
- mixture temp is lowered
- allows primers to bond to the complimentary ends of DNA via hydrogen bonds
outline elongation
- optimum temp for Taq polymerase
- catalyse the free floating DNA nucleotides which join together via complimentary base pairing
- this forms a double strand of DNA
what do PCR tests show?
exponential increase of DNA fragments
what does a curve on the PCR graph suggest?
- it increases as DNA doubles at each cycle
why would a PCR graph plateau off?
- as there are limited primers available within the solution, which will limit how many DNA fragments can be made
