PCR primer: 2 restriction sites? - (Nov/22/2005 )

This gets a little complicated, I realize, so I will just state my question in simplified form at the beginning of the post... How long is too long a non-annealing 5' overhang on a PCR primer (21 bp annealing)? If anyone wants to wade into the rest of the post, I welcome you to!

I am using PCR to make insert to clone into a gene already in pcDNA3.1. The area of the gene I am putting this insert is an intron, and poor in restriction sites. To make the insert, I will be doing PCR from another plasmid, and the insert will be quite long (5 kb). I was just wondering, is it ok to put two non-annealing restriction sites at the 5' end of one of my primers? I need to add another unique restriction site for further cloning once of I have the long insert cloned. Will a very extensive stretch (18 bp!) of non-annealing sequence cause problems during PCR? How big a difference between the melting point of the primer as a whole vs the part that anneals to my amplicon is tolerable? I am kind of new to cloning, so I'm not really sure what I can get away with.

Chuck

PS, my other alternative is to use the original restriction site (MfeI) for the next cloning step, but since it will already exist at the 3' end of the insert I would then have to do an incomplete digestion, which seems more difficult. (And I can't use EcoRI at the 3' end of the insert since there are multiple EcoRI sites within my insert). Anyway, this is a little complicated.

It should be no problem. Your primary concern will be how effective the restriction enzyme is at cutting the ends of the double-strand. To be certain, leave four to six bases (that aren't part of the recognition sequence) at the ends of the primer or check the back of the NEB catalog to see how efficient your enzymes are at cutting at the very end. You could also clone it into a TOPO vector and then cut it out with the appropriate restriction enzymes to prevent this from being a concern.

-Matt

thanks a lot. I checked that table to make sure I am leaving enough bases at the end.

Chuck

Yeah this should work ok, subject to the warnings MisticMatt pointed out -- also leave enough room between the adjacent enzyme sites so they'll both cut...

I once put a restriction site and a promoter region (analogous to E. coli's TTGACAN{16,18}TATAAT promoter) on the 5' end of a primer; it was like thirty-seven or thirty-eight 5' bases and it worked okay.

Remember that as your primer length goes up, the amount of full length primer synthesized goes down...

I agree, this should work fine. I have switched now to always adding the sequence 5' GTTTCT 3' to the 5' end of my primers when I need to have overhang of restriction sites. This is the concensus sequence for encouraging additon of A's by Taq, useful for TA cloning. Since sometimes TA cloning and cutting the fragment out of the cloning vector works better than cutting the PCR fragment directly, this approach optimizes both possibilities at no extra cost or effort.