Design a primer pair each 8 nucleotides long to amplify the

Design a primer pair, each 8 nucleotides long, to amplify the following hypothetical gene through PCR (assume it is double stranded DNA, with only 1 strand shown): 5’ATGCAATCAGCTATTGCTAACAAAGCCGCTATAGGATACAAGCAATTAGAGGAA3’

Solution

Writing down the complementary strand,

5\' ATGCAATCAGCTATTGCTAACAAAGCCGCTATAGGATACAAGCAATTAGAGGAA 3\'

3\'TACGTTACGTCGATAACGATTGTTTCCGGCGATATCCTATGTTCGTTAATCTCCTT 5\'

In PCR two primers are used forward and reverse. A primer is usually 18-20 bp in length to get a good binding before the DNA polymerase begin the replication. A primer has a free 3\' end since the DNA polymerase has a 5\' to 3\' polymerase activity, it needs a free 3\'OH to extend the primer DNA and replicate the parent strand.

Reverse primer

5\' ATGCAATCAGCTATTGCTAACAAAGCCGCTATAGGATACAAGCAATTAGAGGAA 3\'

DNA replication<-------3\'ATCTCCTT5\'

Forward primer

3\'TACGTTACGTCGATAACGATTGTTTCCGGCGATATCCTATGTTCGTTAATCTCCTT 5\'

5\'ATGCAATG3\'-------------> DNA replication

It is usually preferred to design a primer with a G or C base as a last base since G-C binding is more stable and strong due to 3 hydrogen binds than A-T double hydrogen bonds.