Why the addition of restriction sites to primers? - (Aug/22/2008 )
Dear all,
Why would one add restriction sites to their primers? From the nature of primers, it'll bind only to complementary bases. Say, if the sites for EcoRI (that is GATTC), then the primer will only bind to sequence having such bases. If it does bind, the other bases in the primer after GATTC may not bind and what's their use?
Or did I understand correctly the principles of adding restriction sites to primers?
Why would one add restriction sites to their primers? From the nature of primers, it'll bind only to complementary bases. Say, if the sites for EcoRI (that is GATTC), then the primer will only bind to sequence having such bases. If it does bind, the other bases in the primer after GATTC may not bind and what's their use?
Or did I understand correctly the principles of adding restriction sites to primers?
When you add a restriction site, it is only a small part of the whole primer (6 bases out of 20-25).Your primer will initially anneal most readily to your DNA of interest (because there is more complimentary sequence there) and the restriction site will behave like an overhang. During the first round of PCR the complimentary strand of the overhang will be added, and in subsequent rounds of PCR this site will be amplified along with the rest of your PCR product. In the end, you will have made your DNA of interest plus restriction sites on both ends that you can now cut and ligate into an appropriate site.
Why would one add restriction sites to their primers? From the nature of primers, it'll bind only to complementary bases. Say, if the sites for EcoRI (that is GATTC), then the primer will only bind to sequence having such bases. If it does bind, the other bases in the primer after GATTC may not bind and what's their use?
Or did I understand correctly the principles of adding restriction sites to primers?
When you add a restriction site, it is only a small part of the whole primer (6 bases out of 20-25).Your primer will initially anneal most readily to your DNA of interest (because there is more complimentary sequence there) and the restriction site will behave like an overhang. During the first round of PCR the complimentary strand of the overhang will be added, and in subsequent rounds of PCR this site will be amplified along with the rest of your PCR product. In the end, you will have made your DNA of interest plus restriction sites on both ends that you can now cut and ligate into an appropriate site.
Wow, thanks smu2. Frankly, I never know that. To ensure ligation of this fragment into a vector, I'd need the restriction site both in my forward and reverse primer so that both end of my fragments have the sticky/blunt ends. That said, is a double digestion/ligation possible with only 1 amplified fragment?
You should generally aim to use two different restriction sites in your forward and reverse primers so that you get unidirectional ligation into your vector because otherwise you will end up with clones having incorporated your insert in two different orientations (same holds for blunt end ligations). Another problem is that your vector will religate if only cut with one restriction site, therefore the efficiency of cloning will be greatly reduced unless you dephosphorylate it prior to ligation. When you design your primers make sure you have enough bases outside the restriction site so that it can actually be bound and properly cut by your enzyme.