Bio 2c03: Polymerase chain reaction week 12-13 Flashcards
What does PCR use?
It uses dna polymerase which is an in vitro replication of specific dna sequences
What does pcr generate?
Can generate tens of billions of copies of a particular dna fragment from a dna extract
What is TAQ?
A dna polymerase which serves as a standard reagent for pcr reaction
At what temperature is TAQ stable? why is this useful?
95 degrees, extreme temperatures are needed to make it go from double stranded to single strand
What is the processivity of Taq polymerase?
50-60 nucleotides (nt) per second at 72 degrees celsius.
What are the two primers used for a polymerase chain reaction? how long are they?
forward and reverse primers- 18-30 nucleotides in length
What do primers do?
Primers are no longer than 50 nucleotides and determine the beginning and end of the region to be amplified; the polymerase synthesizes the complementary sequences from each primer
What do the primers depend on?
- primer length
- melting point
- specificity
- complementary primer sequences
What is the processitivity for the extension step for taq
1000 nt per minute
What is the fidelity of taq?
Low fidelity because it is not accurate, leads to mutations, suitable for 500 base pairs or less.
When should high fidelity dna polymers be used?
should be used for experiments involving mutation detection and dna sequencing
What are the 3 main processes involved in polymerase chain reaction?
denaturing/melting, annealing, elongation/extension step
What is the denaturing/melting step
Tax polymerase used, temperature increased to 94-96 degrees for 10-20 mins, causing 2 complementary strands
What is the annealing (hybridization) step?
the temperature is lowered 2-5 degrees from the melting/denature temperature, and hence the primers attach to single dna strands
What happens in the extension step for a polymerase chain reaction?
The dna polymerase fills the missing strands; the temperature involved will be dependant on the processivity of taq polymerase
What is the temperature involved in the extension step for taq polymerase and how long does it take to start?
72 degrees, takes 5-15 minutes
How is the application of dna polymerase done?
on a logarithmic scale
What is the amplified product of the pcr process called?
an amplicon
How many different types of polymerase chain reactions are there? name 2 important ones
roughly 31 so far, two important ones are multiplex pcr and nested-semi nested pcr
What is multiplex pcr?
used to amplify several targeted regions in a single reaction
Why is the design for primers for each region in a multiplex pcr different?
It uses different primers so they can stick to different parts of the dna that needs to be amplified
What are the drawbacks of multiplex pcr?
- the melting temperature of the primers has to be close enough to anneal at the same annealing temperature
- the base pair lengths should be different to form distinct bands because varying sizes of different genes are targeted
What is nested-semi nested pcr
it involves amplifying a larger sequencing and then amplifying a smaller part of that large sequence
How many primers are used for a single locus point for nested-semi nested pcr?
Two primers are used for a single locus point
