Primer Design Step by Step

1.- Select the aminoacid positions that you want to mutate according to the crystal structure or homology model of the protein. NOTE: In the absence of crystal structure or homology model you should in principle create error prone PCR libraries at high mutation rate, due to the fact that it is not possible to do rational design in the absence of a reference model.

2.- Once you know exactly which positions you want to mutate or randomize, you just need to identify the codons that codify those amino acids in the gene encoding sequence.

3.- The primers should be designed following the recommendations of the mutagenesis method that you want to use, QuikChange, Megawhop, Multi-QuikChange, Overlap extension PCR, etc.

4.- a) To design site-directed mutants i.e. an specific amino acid change e.g Leu162Ala you just have to change the codon by the new desired codon taking into account the codon usage of the strain you want to use for expression. b) to design site-directed libraries, you need to change the codon by a codon degeneracy, for example NNK, that encodes all amino acids, although with bias, that is to say that there are overrepresentation of some amino acids, NDT or NHT that encode 12 amino acids, covering all chemical groups, and just with one codon per amino acid. Using CASTer (Reetz Group) or the mini-lib design tool (Mena and Daugherty) you can select different codons and amino acid combinations and evaluate the "screening effort" in terms of number of colonies to be screened.

I. Primer Design for Quick Change using Invitrogen Vector NTI

1.- Open Vector NTI

2.- Open the plasmid/gene that you want to work with. In this example pTRC - Bacillus subtilis lipase.


3.- Select the gen that encodes the enzyme

4.- In the sequence Window press the right button of the mouse over the DNA sequence of the gen (selected) and clic over "Translate Direct Strand" to get the aminoacid sequence encoded by the DNA.

You obtain the aminoacids encoded by the DNA codons


5 .- You have to find "by hand" the aminoacids that you want to mutate. In this example we will make a library in positions Ile12 and Gly13 using the degenerated codon DBK. The manual way s to take into account the number of base pairs selected in blue (you can see it directly in the botton of the window). Each aminoacid is encoded by one codon i.e. 3 bases, so 33 bases will encode 11 aminoacids, so the next codon will correspond to the Ile 12 that we want to mutate.

[DESIGN PRIMERS FOR MEGAWHOP]

6.- DESIGN PRIMERS FOR QUICK CHANGE. According to the QuikChange manual, we have to select around 15 base pairs at each side of the 2 positions that we want to mutate.

7.- Once selected (highlighted in blue) clic at List/Add Selection to Oligo List

8. Give a name to the primer (this primer will be the forward primer)

9. Open the Oligo List

10.- Insert the mutations. We want to change Ile12, encoded by ATT to DBK and Gly13 encoded by GGA to DBK. To do it we edit the oligo in the Oligo List.


11.- Analyze the Oligo to see if accomplish the characteristic demanded for Quik Change.

This primer has a GC content of 46% that is higher than the 40% suggested in QC manual.
The melting temperature of 77 ēC is almost the 78 ēC proposed in the manual, and according to our experience the QC works even with lower melting temperatures of the primers.

Palidromes are sequences of bases that are the same whatever the sense you read them. e.g. AGACTTCAGA. If the palindromes are large and with a high G-C content the primers may anneal in the non desired orientation.

Repetitions are not so important when they are less than 5 bases, and when the repetitions involves triple bond former bases (G,C) are specially important. Sometimes itis not possible to avoid this when designing Quick Change (QC) primers and sometimes the option of using megawhop should be taken in to account. But in general lines QuikChange works always, only plasmids with very high G-C content have presented problems.

12.- Creation of the reverse primer

Copy the sequence from the forward primer with using CTRL+C or the right buttom of the mouse, then go to the oligo list and press "Add".

Paste the Oligo that you have copied

then press "Reverse complementary"obtaining the reverse primer, that already encodes the degenerated codon desired.

Press Accept and you have both primers already designed.

13.- Check if the primers are able to anneal in other region of the plasmid/gene.
For that we should clic over Analyses/Motifs and select the oligo-list as source of the sequences.

We press Oligo List to get the oligonucleotides that we have designed and then OK. Then in the left Window of Vector NTI we can see a folder called Motifs.

As we see both primers are only annealing in (1 site) as desired. The percentage of similarity with the native sequence depends of course of the amount and the degeneracy of mutations that are implemented in the primers. The primers appear as well annealed in the DNA sequence viewer.

14. Save the Primers in the Database. (You should do it one by one)

If you do it this way, it in general works fine, but you can also optimize it a bit more considering general recommendations for primer design:

- primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming.
- 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;
- primer self-complementarity (ability to form secondary structures such as hairpins) should be avoided;
- runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.

15. ORDERING THE PRIMERS

Select the primers in the oligo list and clic over the Invitrogen Icon. You will be redirected to invitrogen (if you are not redirected check the Security Level of Internet Explorer or the Pop-up blocking Properties).

 

The desalted primers (cheaper and fast delivered) are OK for normal QC of 1 or 2 positions if you want to add randomization in 3 or more codons is better if you select the HPLC purified ones. Is not needed any kind of 3' or 5´ modification.

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