Khan Academy Recombinant DNA

[663697]

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Restriction site:

Gene of interest:

This question has been giving me some trouble. Can somebody please explain where to start? Having a hard time picturing where everything goes..

 

[Nugester]

The question is asking you to design a reverse primer that has the Sall RE site on the end and remember, all primers need to be 5' -> 3'. This means 5'-GTCGAC..." should be the beginning. So you can elimiate A and C based on that, leaving B and D. Between B and D, part of the gene of interest needs to be there, starting at the 3' end of the gene of interest since PCR synthesis is from 5'->3'. Complementary to AATCCCGAC is TTAGGG. So the primer would be 5'-GTCGACTTAGGGCTG. This eliminates B and leaves D. You don't have to worry about the extra TTA in the primer because it isn't a part of the gene of interest, similar to Sall.

 

[663697]

The question is asking you to design a reverse primer that has the Sall RE site on the end and remember, all primers need to be 5' -> 3'. This means 5'-GTCGAC..." should be the beginning. So you can elimiate A and C based on that, leaving B and D. Between B and D, part of the gene of interest needs to be there, starting at the 3' end of the gene of interest since PCR synthesis is from 5'->3'. Complementary to AATCCCGAC is TTAGGG. So the primer would be 5'-GTCGACTTAGGGCTG. This eliminates B and leaves D. You don't have to worry about the extra TTA in the primer because it isn't a part of the gene of interest, similar to Sall.

Ohhh so the Sall site of the reverse primer (going 3'<-5') binds to 6 base pairs to the right of "5' ...TAA 3' " of the gene of interest? So you would have 3'.....ATT(CAGCTG)5' as your reverse primer with CAGCTG binding outside of the gene of interest so that the gene of interest is amplified? In that case what's the point of them using the sequence of Sall as their primer? Do they know that the gene of interest has that same restriction site somewhere downstream for the Sall primer to bind to?

 

[Nugester]

Ohhh so the Sall site of the reverse primer (going 3'<-5') binds to 6 base pairs to the right of "5' ...TAA 3' " of the gene of interest? So you would have 3'.....ATT(CAGCTG)5' as your reverse primer with CAGCTG binding outside of the gene of interest so that the gene of interest is amplified? In that case what's the point of them using the sequence of Sall as their primer? Do they know that the gene of interest has that same restriction site somewhere downstream for the Sall primer to bind to?

They are using the restriction site because they want to put into something, such as a plasmid or vector. Then you can grow it. For example, bacteria that takes in the gene, divides rapidly and amplifies the gene of interest.