Question

Design primers for the following target sequence which is to be amplified using PCR:

CGGCTATCAGGATTGTTCTCTGGACCGTACATCGAACGGCTATCGAGGATTGTTCTCTGGACCGTACATTAAA

Explain how you went about designing them.

Answer 1

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To design a primer one must take care that the primer should be compatible with the target DNA should have the the appropriate amounts of nucleotide content and also should have a start code. A primer should be typically of length 17 to 18 bp. There are several other rules apart from these.

Consider the given sequence is from 5 prime to 3 prime strand of a a double stranded DNA. Now now a forward primer and reverse primer are required to carry out the PCR reaction. The forward primer is usually attached to the the complementary strand of the given strand so the primer should be the same sequence as the given stand. In this case the primary would be

5' CGGCTATCAGGATTGTTCTCT 3'

The extension always happens on the three prime end of the primer.

And for the reverse primer the primary should be attached to the given strand starting from the last nucleotide. So here primer should be e the reverse sequence of complementary of the given sequence.

5' TTTAATGTACGGAGAGAACA 3'

SO NOW BOTH THE FORWARD AND BACKWARD PRIMER ATTACHED TO BOTH THE ENDS OF THE DOUBLE STRANDED DNA AND START EXTENDING TOWARDS THE OTHER SIDE.