We hope that the first Tips and Tricks was helpful, but that just scratched the surface of the power of PCR.   Keep reading for more advanced techniques that will allow you to exploit PCR for everything it is worth.  We welcome the opportunity to share these with you. 

It's Tips and Tricks Tuesday!

Everyone knows that PCR is a great way to amplify DNA, but with careful design of the oligos you can add mutations of all kinds.  For example, point mutations are best added in the middle of the oligo (if possible), but larger mutations, such as sequence to add an expression tag (like the 6x His tag) should be part of the 5’ end of the oligo.  In fact the 5’ end of the oligo can have a great deal of flexibility in its sequence, as long as the 3’ end has perfect matching sequence.

Remember though, when introducing mutations, the Tm of the oligo will change.  At first, the oligo will anneal only to the 3’ end with the perfect match, but, after a few rounds, there will be newly synthesized template that has incorporated the mutation oligo, so the annealing will now be the entire sequence of the oligo, not just the 3’ end.  You can optimize your PCR conditions by running a 2 phase run.  For a few rounds (5-10) assume that only the matching 3’ end of the oligo will anneal and calculate the Tm accordingly.  After that, switch the annealing temp (or switch to a 2 step PCR if the oligos allow it) to take into account the higher Tm of the entire oligo annealing.

Single point mutations are useful, but you can use the same concept for making libraries of different sequences. For example, a common tactic is to replace a particular amino acid in a protein with every other option (so 20 different possibilities -19 mutants and 1 wild type).  You don’t have to synthesize 20 different oligos to do that, thanks to the degeneracy of the genetic code (and some creativity) you can get all 20 codons with about 7 different oligos (even fewer if you eliminate certain amino acids that are likely to be really problematic like proline).

Hit us up for the list of 7 sequences that hit every amino acid codon 1 time each.

We are happy to help!

Just like you can add mutations or “tags” to the ends of the sequence, you can also add restriction sites to the ends of the sequence for directional cloning into your vector. Remember to add 6 bases between the end of the oligo and the new restriction site so you can digest the PCR product and ligate directly (versus cloning the product into a TA, or similar, vector first).  Even if you purify the product, digestion at the ends of PCR products can be inefficient so digest longer than you normally would to ensure sticky ends.  Otherwise it will act like any other vector/insert ligation (and save you several days of sub cloning and shuttling from one vector to another).

Larger mutations (dozens of bases) can be introduced into coding sequences without a local restriction site through PCR.  This will require a few steps and can get complicated.  Send us a message if you need clarification.

Basically, you will set up multiple PCRs.

1. From the 5' end (using  a convenient restriction site as a starting point-see tip 3) to the region to be mutated.
2. From the area to be mutated (where PCR 1 ended) to the 3' region of the final desired sequence (typically terminating with a different restriction site).  Note the antisense oligo from PCR1 and the sense oligo of PCR2 will be reverse compliments of each other.
3. Run the 2 PCRs and collect the fragments.  The 3' end of PCR1 will have the same sequence as the 5' end of PCR2.
4. Mix the two fragments and add the sense oligo from PCR1 and the antisense oligo from PCR2 in PCR3.  The two fragments will hybridize with their identical sequence and fuse together into 1 product.
5. The final product of PCR3 will be your final sequence with the mutations in the middle.

This method can be used to fuse any sequences together, so get creative.  You can also repeat the same technique to fuse more than 2 sequences together, just repeat the process for each pair and then link together.  Try to reduce the number of separate PCRs (to limit mutations) by doing pairs together and then fusing those products, rather than one at a time.

SO if you are going to put 4 pieces together, then fuse 1 and 2 together and fuse 3 and 4 together, then fuse 1-2 with 3-4.  It is also possible to put together more than 2 pieces at a time, but the optimization gets much more difficult.  Doable, but increasingly harder.

Finally, you can integrate any insert into any vector by PCR (without any restriction sites). It takes a bit of planning up front.

• First, decide if you are going to fuse the 5’ or 3’ end of the PCR product (assume the 5’ end will be fused for this example, but it doesn’t matter really).
• Second, determine the point where the insert will be fused into the vector (this can be basically any point).
• Third, design a sense oligo to the vector, starting at the insert point and going 3’ away from that point
• Fourth, design an antisense oligo to the vector starting at the insert point and going 5’ away from that point (I know that sounds confusing but imagine you are making a PCR product of the entire vector so that it is linearized at the insert point) This oligo has to have complimentary sequence to the 5’ section of the insert.
• Fifth, design a reverse compliment oligo (sense) to the one you made in the fourth step.
• Sixth, amplify the vector with the oligos from the third and fourth steps (this will be a long PCR depending on the size of your vector). Amplify the insert with the oligo from the fifth step and the 3’ oligo from however you made the insert.
• Seventh, mix the two PCR products and then fuse them together like you do in tip 4. This produces a linear piece of DNA that has the vector and insert attached at the insertion point.
• Finally, purify the product and add ligase. The intra (within one molecule) ligation is preferred over the inter (between two molecules) ligation so you will end up with nicked circles.  Even if you don’t phosphorylate the ends first, this works as the bacteria will repair the nicks once you transform them.  It sounds complicated, but it is really just an extreme version of tip 4.