CHAPTER 2 (PRINCIPLES IN PCR TECHNIQUES)

Question 1

what is the three step cycle of PCR?

Answer 1

denaturation (heating), annealing (cooling), extension (replication)

DAE

Question 2

definition of denaturation

Answer 2

the process where the double-stranded DNA is separated into single strands by breaking the hydrogen bonds between the base pairs. This typically happens by applying heat (usually around 95-96°C), though other factors (like pH or chemicals) can also cause denaturation.

Question 3

wjat does the Tm of DNA depends on?

Answer 3

GC content. higher GC higher Tm

Question 4

definition of renaturation

Answer 4

process where single-stranded DNA (or RNA) re-forms a double helix by reforming hydrogen bonds between complementary base pairs. This occurs when the temperature is lowered (usually to around 54°C) or when the denaturing conditions are removed, allowing the strands to reassociate in their correct complementary sequences.

Question 5

time and temp of the three step cycle in PCR

Answer 5

1. denaturation: 94C for 2 mins (initial), 15 sec in step 2)
2. annealing: 50-65C for 20 sec
3. extension: 72C for 1 min per 1kb

Question 6

PCR produces an ____ population of ____

Answer 6

1. exponentially growing
2. identical DnA molecules

Question 7

formula to find number of copies made in PCR

Answer 7

final DNA copies= initial copies X 2^number of cycles

assuming 100% efficiency (which is rare)

Question 8

definition of thermal cycling

Answer 8

process used in PCR where the temperature is repeatedly changed in a series of steps to denature, anneal, and extend DNA. These cycles typically involve three main temperatures:

1. Denaturation (94C) – separates the DNA strands.
2. Annealing (~50-65°C) – allows primers to bind to the DNA.
3. Extension (~72°C) – the DNA polymerase synthesizes the new DNA strand.

This process is repeated multiple times to amplify the DNA.

Question 9

what does PCR requires (ingredients)

Answer 9

1. buffer (+MgCl2)
2. dNTPs
3. DNA polymerase (Taq polymerase)
4. oligonucleotide primers
5. DNA sample (target DNA)

and thermocycler