SAC 1? - PCR Flashcards
(10 cards)
1
Q
examples of why we need to amplify DNA
A
- forensic testing (bodily fluids)
- paternity testing
- analysing gene fragments for genetic diseases
- testing for infectious disease
2
Q
main steps and temperatures in PCR
A
- denaturation (95ºc)
- annealing (55ºc)
- elongation (72ºc)
- repeat
3
Q
denaturation
A
- hydrogen bonds broken and DNA splits into 2 strands
- done bc primer needs to be added to one singular strand
- 90-95º
4
Q
annealing
A
- primers (short pieces of DNA) bind where complementary sequences are (3’ end)
- done so Taq has a starting point/defines region of DNA to be amplified
- 50-55º
5
Q
Elongation/extension
A
- Taq binds to primers, moves along each strand
-adds free nucleotides to form new, identical DNA (double stranded) - 72º because it is the optimal temp for Taq
6
Q
necessary ingredients for strand synthesis
A
- DNA sample
- Taq polymerase
- additional nucleotide bases (A, C, G, T)
- forward + reverse primers
7
Q
explain the role of the primers
A
- short segments of complementary DNA that join to 3’ end of single stranded DNA
- Taq attaches to them and begins extending
- define the region of DNA that will be amplified
8
Q
what are the 2 primers and why does PCR need 2?
A
2 primers: forward and reverse - bind to the double-stranded DNA molecule that has been separated into 2 single strands
- FORWARD: binds to 3’ end of TEMPLATE strand -> Taq synthesises strand in 5’-3’ direction (forward)
- REVERSE: binds to 3’ end of CODING strand -> Taq synthesises in 5’-3’ in reverse
9
Q
What is Taq polymerase?
A
- polymerase enzyme required in elongation stage
- binds complementary nucleotides together when copying single-stranded DNA
10
Q
Why is Taq polymerase the enzyme used in this process?
A
- it is heat resistant as it is isolated from bacteria that live in hot springs
- can withstand extreme temperature changes required by PCR
