PCR

Question 1

What is PCR

Answer 1

To amplify a small length of DNA into numerous identical copies

Question 2

What is the purpose of PCR

give examples

Answer 2

Forensic Testing
Paternity testing
Detecting mutations

Question 3

What is a primer

Answer 3

A primer is designed as a short single stranded DNA sequence that acts as a starting point for DNA synthesis

Question 4

What do you put in the PCR machine?

Answer 4

.DNA sample
.Taq polymerase
.Nucleotide bases
.DNA primers
MgCl2( it is a cofactor for DNA polymerase)

Question 5

Why is there a buffer to maintain the pH level?

Answer 5

Because as number of Deoxyribose nucleic acid increases the more the solution gets acidic, which then the Taq polymerase might not be working best in different pH levels.

Question 6

What is the relative tempreature for Denaturation step?

Answer 6

90-95c

Question 7

What happens in the Denaturation step

Answer 7

The DNA is heated and breaks the hydrogen bonds, making single stranded molecules.

Question 8

What is the relative temp for Annealing step

Answer 8

50-55c

Question 9

What happens in the anealling step

Answer 9

The DNA strands are cooled allowing primers to bind to the complemantry sequences in the DNA.

Question 10

What relative tempreture is used for the Elongation step?

Answer 10

72C

Question 11

What are the exact steps for PCR?

Answer 11

1. Denaturation, DNA is heated to 90c-95c to break the hydrogen bonds between the double strands to make them single stranded.
2. Annealing The single stranded DNA is cooled down to 50c-55c to allow the primers to bind to its complementary sequences.
3. Elongation Dna is reheated to 72C, for the enzyme Taq polymerase to work in optimal conditions. TAQ polymerae binds to the Primers and starts to make a new complementary strand of DNA.
4. Repeat Repeat the PCR cycle repeatdly.

Question 12

What is formula for the number of DNA (DOUBLE STRANDED) is created per PCR cycle?

Answer 12

x= 2^n

two to the power of the pcr cycle number

Question 13

Why does the phosphate backbone not break during the first step of PCR which is?

Answer 13

Denaturation step breaks H bonds because it is weaker than the covalent bonds on the phosphate backbone.

Question 14

Primers are often designed specifically for annealing by synthesising it. TRUE OR FALSE?

Answer 14

true

Question 15

What are the two primers needed for the coding strand and template strand?

Answer 15

Foward primer for template strand and Reverse Primer for coding strand

Question 16

What does the foward primer do

Answer 16

It binds to the start codon at the 3’ end of the template strand. TAQ polymerse then makes the strand the same direction RNA polymerase creates mRNA

Question 17

What happens to enzymes if the tempreture was raised and decereased

What to mention in the answrr

Answer 17

Talk about the relation to rate of reaction
The enzyme denatures only past optimal
Talk about confirmational shape change in active site
Talk about if lowered temp, there wont be enough kinectic energy

Question 18

Why do you need two different primers in PCR

Answer 18

You need two different because the complementary sequences are differnet at 5’ end of the coding and template strand.

Question 19

When do you use PCR for and when you do use gene cloning for

Answer 19

PCR is used when small samples of DNA is available for labortaotry testing/forensics whereas Gene cloning is used to make copies of larger sections of DNA, whcich is used to insert DNA into a cell and produce a protien product in large quantaties.

NOTE: PCR is much quicker compared to gene cloning.