Question

5’-GGGGGATGTCGACTTGATCTGATATCGGGGGTGTGATATCTGCAAAGATCTGCTATTTT-3’ 3'-CCCCCTACAGCTGAACTAGACTATAGCCCCCACACTATAGACGTTTCTAGACGATAAAA-5' Your colleague wants to amplify the above sequence and he has designed the following primers to correspond to the ends of the 60 base pair target sequence A) 5’—GGGGG—3’ and B) 5’—AAAATAGCA—3’ Your colleague decides to synthesize the sequence using Gibson assembly as opposed to amplifying with PCR. Starting with the primer below, provide the sequence of additional 10-base pair long primers required to synthesize the total 60 bp region. For this question, all primers have to be 10 bases long and you can assume you need 5 base pairs of overlap between primers. (8 pts) 5’-GGGGGATGTC-3'

Answer 1

In the Gibson assembly the base of the primers overlap so that they could be linked to form the entire strand of DNA.

therefore the Primers should be like this:

5’—GGGGG—3’, 5'- CCCCCATGTC- 3' 5'- TACAGCTGAA - 3' 5' - GACTTCTAGA - 3'

and B) 5’—AAAATAGCA—3’ 5' - ATCGTGATCT - 3' 5'- CTAGATTGCA - 3'

We would design it in the same way for the entire 60 base pair sequence.