You wish to amplify the following double stranded DNA segment using PCR. The fol

Question

You wish to amplify the following double stranded DNA segment using PCR. The following sequence represents one of the two strands of the duplex molecule, which has an antiparallel complement (not shown). What are the sequences of the 2 oligonucleotide primers (5' to 3') that you would use to amplify this entire DNA segment? Note that the Tm values of the primers should be similar.

5'- ACGTGCCTCTCCTCATACGAGGATATATATAGTGTTATTTTGTTTGGGGTGTTTGTTTTGGATTGT
GTTTTTTTGTTTATTTCGTGTGTTATGTAACATATGACGTGCCTCTCCTCATATAATTTCTATCTACTT
GCACAATGAAATATATTCATAAAGCATAAGCTCATTG-3'

Explanation / Answer

Primers would be added to the 3' end of the dsDNA. Also, the length is 18-20 base pair.

Primer for the one mentioned in question would be- 5' CAA TGA GCT TAT GCT TTA .

For the complementary sequence primer would be-

DNA strand 3' CTC ATA CGA GGA TAT ATA

Primer 5' GAG TAT GCT CCT ATA TAT.

We need to maintain the GC % in both the primers to have similar Tm value. The primers thus selected above both have 33% GC content. In the complementary DNA sequence (to the sequence mentioned in question), primer is selected complementary to nucleotides leaving few nucleotides from the start.

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