Question

You are performing a simple polymerase chain reaction (PCR) to amplify ANY 300 - 500 bp region (for example nucleotide 5 to 555 or 20 to 575). Using your primers, you will run a theoretical PCR reaction, and then run the product on an agarose gel to determine if your PCR reaction worked correctly.

CAGGAAACAGCTATGACCATGATTACGCCAAGCTTGGTACCGAGCTCGGATCCACTAGTA

ACGGCCGCCAGTGTGCTGGAATTCGGCTTCCCGGAGCCGGACCGGGGCCACCGCGCCCGC

TCTGCTCCGACACCGCGCCCCCTGGACAGCCGCCCTCTCCTCCAGGCCCGTGGGGCTGGC

CCTGCACCGCCGAGCTTCCCGGGATGAGGGCCCCCGGTGTGGTCACCCGGCGCGCCCCAG

GTCGCTGAGGGACCCCGGCCAGGCGCGGAGATGGGGGTGCACGAATGTCCTGCCTGGCTG

TGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCA

CGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAG

AATATCACGACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGAC

ACCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTC

TGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGGTCAAC

TCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCAGTGGCCTTCGC

AGCCTCACCACTCTGCTTCGGGCTCTGCGAGCCCAGAAGGAAGCCATCTCCCCTCCAGAT

CAGCCTGTCCCATGGACACTCCAGTGCCAGCAATGACATCTCAGGGGCCAGAGGAACTGT

CCAGAGAGCAACTCTGAGATCTAAGGATGTCACAGGGCCAACTTGAGGGCCCAGAGCAGG

AAGCATTCAGAGAGCAGCTTTAAACTCAGGGACAGAGCCATGCTGGGAAGACGCCTGAGC

TCACTCGGCACCCTGCAAAATTTGATGCCAGGACACGCTTTGGAGGCGATTTACCTGTTT

TCGCACCTACCATCAGGGACAGGATGACCTGGAGAACTTAGGTGGCAAGCTGTGACTTCT

CCAGGTCTCACGGGCATGGGCACTCCCTTGGTGGCAAGAGCCCCCTTGACACCGGGGTGG

TGGGAACCATGAAGACAGGATGGGGGCTGGCCTCTGGCTCTCATGGGGTCCAAGTTTTGT

GTATTCTTCAACCTCATTGACAAGAACTGAAACCACCAAAAAAAAAAAAAAAAGCCGAAT

TCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGC

CCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAA

TCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCAGTGGCCTTCGC

AGCCTCACCACTCTGCTTCGGGCTCTGCGAGCCCAGAAGGAAGCCATCTCCCCTCCAGAT

GCGGCCTCAGC

Questions related to scenario:

1. Design your forward and reverse primers, following the guidelines.
   1. Primer length 15-30 nucleotides
   2. Tm should range between 55 – 65C
   3. Tm of primer pair should be within 5C of each other
   4. GC content of primer = 40 – 60%
   5. Try to avoid a row of 4 or more of the same bases so for eg GGGGG or ATATATATAT
   6. 3’ end of primer should end with G or C

2. Provide the sequences in a 5’-3’ direction as if you were going to order them. Include the start and stop bases and number for both primers and length of primer.
   1. Forward primer in 5’ – 3’ direction.
   2. Reverse primer in 5’ – 3’ direction.

3. Estimate the melting temperature of the primers you designed. Tm =4(number of GC) + 2(number of AT)

4. Describe the thermal cycling protocol that you would predict to work. Provide the denaturation temperature, the annealing temperature, and the extension temperature.

Answer 1

a)Forward Primer=5'ATGACCATGATTACGCCAAG3'

Tm=4(9)+2(11)=36+22=58C

Melting Temperature=58C

Annealing temperature =53C

Denaturing Temperature =92C

Extension Temperature =72C

Thermal Cycling protocal=DNA denatures at 92C and when cooled to 53C it starts to reannealing Primer attaches to the DNA and starts the reaction.

b)Reverse primer=5'-ACAGCTATGACCATGATTAC-3'

Tm=4(8)+2(12)=32+24=56

Melting Temperature =56C

Annealing Temperature =51C

Denaturing Temperature =92C

Extension Temperature =72C

Thermal cycle protocol =DNA denatures at 92C when it is cooled to 51C Primer attaches to the DNA and starts the reaction.