Question

You are provided with two primers below and you wish to amplify the CYC1 terminatoron pMD1:

Primer 1: CTATGAAACAAGAATCGACC

Primer 2: GTGCCGTAAAGCACTAAATC

a. What secondary structure is formed by each primer at 54°C?

b. What secondary structure is formed by both primers together at 54°C?

c. If we were to use the following PCR protocol with primer 1 and primer 2, what would we expect to see on the electrophoresis gel?

Step	Time	Temperature
Initiation	2’	95°C
Denaturation	1’	95°C
Annealing	1’	53°C
Extension	1”	72°C
Final extension	10’	72°C
End	4°C	∞

d. Suggest an optimized PCR protocol based on the one from question 24 and using primers 1 and primer 2. Explain also how it would improve somewhat the amplification of the CYC1 terminator.

Step	Time	Temperature
Initiation
Denaturation
Annealing
Extension
Final extension
End

Answer 1

A) primer 1 separately may form self dimer and primer 2 forms hairpin in 54°c

B) heterodimer may be formed with both primer together

C) due to secondary structures , there will be less amplification in PCR

D)

Step	time	temperature
Initiation	2	95
Denaturation	1	95
Annealing	1	54
Extension	1	72
Final extension	10	73
End	4°	Infinity

By increasing the annealing temperature , we can reduce misamplification and increase the specificity to the particular gene CYC1 terminator