5 You wish to amplify the entire sequence below where NX equ

5. You wish to amplify the entire sequence below (where NX equals a long DNA sequence that will be amplified, but is not in the primer sequences).

upstream 5’-CCTCAGTGAGTGTGACTCAG(NX)GTGCCCATACATTTAGTGCC -3’

downstream 3’-GGAGTCACTCACACTGAGTC(NX)CACGGGTATGTAAATCACGG -5’

5a. Write the sequence of the primers necessary to incorporate a KpnI site on the upstream end and a PstI site on the downstream end of the PCR fragment. Write the sequence of each primer from left-to-right, with the 5’ end on the left and the 3’ end on the right, and label the 5’ and 3’ ends of each primer.

Upstream primer:

Downstream primer:

5b. Write the sequence of the amplified PCR fragment (label the 5’ and 3’ ends of each DNA strand).

5c. Write the sequence of the amplified PCR fragment after digestion with KpnI and PstII (label the 5’ and 3’ ends of each DNA strand).

Solution

5a.

Upstream primer (upstream primer will bind the downstream sequence because it is 3\' to 5\' and primer sequence is given in 5\' to 3\')

5\' GGTACCCCTCAGTGAGTGTGACTCAG 3\'

Downstream primer

5\' CTGCAGGGCACTAAATGTATGGGCAC3\'

5b.

5’-GGTACCCCTCAGTGAGTGTGACTCAG(NX)GTGCCCATACATTTAGTGCCCTGCAG-3’

3’-CCATGGGGAGTCACTCACACTGAGTC(NX)CACGGGTATGTAAATCACGGGACGTC -5’

5c. 5\' GGTACCCTCAGTGAGTGTGACTCAG(NX)GTGCCCATACATTTAGTGCCG -3’

3’-CGGAGTCACTCACACTGAGTC(NX)CACGGGTATGTAAATCACGGACGTC 5\'

5\' GTACGGTACCCCTCAGTGAGTGTGACTCAG 3\'