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I am wondering what the correct method for primer design to introduce restriction sites. Specifically between two methods.

1) Primer first partially hybridises to the gene, has a mis-match where the restriction site and extra bases are on the primer, then has a hybridised site following the restriction site, to DNA upstream of the gene.

or

2) Primer first partially hybridises to the gene, then completely dangling and non-hybridising has the restriction site and extra bases.

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I think the best way is option #2:

Suppose that your gene of interest is AAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGGGGGGGGG

and you want to insert EcoRI restriction site GAATCC

Then your Fwd primer will be GAATCC AAAAAAAAAAAAAAAA

and your Rev primer will be GGATTC CCCCCCCCCCCCCCCC

But in general, it shouldn't matter which option you choose, as long as you calculate the Tm of only the part of the primer that hybridizes to your template.

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The option #2 is most common. Do not forget to add 3 or more additional terminal base pairs for optimal restriction enzyme cutting (source: BioTechniques 1998, 24:582-584)

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