Table of Contents



Introduction

The QuikChange® Primer Design Program allows you to design primers for site-directed mutagenesis (SDM), including both single-site and multiple-site mutagenesis projects. The program suggests primer sequences according to Stratagene's QuikChange kit primer design guidelines and the rules described below.

A primer-template duplex containing mismatches is characterized by having a higher free energy than a duplex without mismatches. In this program, free energy is calculated by the nearest-neighbor method for nucleic acid duplexes. Mismatches in a duplex are treated as internal loops and/or bulges which are flanked by branches of perfectly matched duplex DNA . The total free energy of a primer-template duplex is a sum of base-stacking energies of nearest neighbors plus the energies of loops, bulges, and the terminal "dangling ends." The nearest-neighbor free energy values are taken from the program mfold, version 2.3, and adapted for hybridization.

When designing primers for mutagenesis of untranslated DNA , including nucleotide substitutions, deletions, or insertions, the software designs primers of sufficient length and melting temperature to incorporate the desired mutations.

When amino acid substitutions in a protein sequence are desired, the software selects the most energy-saving base changes to generate the amino acid substitution of your choice. Preference in codon replacement is given to the codon that requires fewer nucleotide substitutions or results in mismatches capable of base-pairing (e.g. G-T). In addition to energy conservation rules, several empirical rules regarding optimal codon replacement were also incorporated into this program, based on Stratagene’s data with degenerate primers1.

SDM Primer Design

Principles of Minimizing Energy Cost

The duplex between the DNA template and the site-directed mutagenesis (SDM) primer is imperfect. That is, it contains one or more base mismatches or single-stranded segments (bulges). Therefore, the free energy of the mismatched duplex is higher than that of a perfect duplex, i.e. a hypothetical duplex between DNA template and perfectly-complementary oligonucleotide. The difference between the free energy of a mismatched duplex and the corresponding perfect duplex is described as the "Energy Cost" of the mismatches. 

                  E0-E1
    Energy_Cost = ----- * 100%
                    E0
where E0 is a free energy of "wild type" duplex and E1 is a free energy of the mutagenesis primer-template duplex.
Primers are designed with the goal of minimizing free energy of the mismatched primer-template duplex, thus minimizing the energy cost. According to the QuikChange primer design principles, the base mismatches must be located close to the middle region of the primer, so that the less stable region of the primer-template duplex is flanked by branches of more stable duplex. If multiple destabilizing base-mispairing events are required, then the flanking stable branches of the primer are made longer to compensate for lowered duplex stability and to minimize the energy cost.

General Guidelines for Using the QuikChange® Primer Design Program Interface

The main QuikChange Primer Design program web interface presents several input controls. We recommend that you fill the form from the top to the bottom, starting with selection of the QuikChange kit that you are using. The DNA sequence (in plain text or FASTA format) can be entered either by selecting a sequence file from a local computer or by typing or pasting the sequence directly into the provided text area.

The sequence can be uploaded either as an untranslated DNA sequence or as a translated protein sequence by selecting the corresponding button. If translation of a DNA sequence is desired, then the optional translation range can be specified. After uploading, the sequence is displayed as a series of checkboxes corresponding to individual polymer residues to allow selection of the desired mutation positions. For point mutations, use the drop-down menus above the sequence checkboxes to specify the type of change needed for each of the selected residues. For deletions or insertions, select the appropriate radio button from below the point mutation drop down menus. After the entire form is filled out, and all necessary parameters are correctly set, submit the design task to the server for validation and processing by clicking the Design Primers button. Results are returned displaying tables with primer names, sequences, melting temperatures, primer-template free energy values, energy cost calculations, and primer-template duplex schemes.

Introducing Multiple Mutations

Multiple mutations, at up to seven regions, can be introduced to both untranslated DNA or protein sequences either by designing a single primer that incorporates multiple point mutations or by creating multiple primers, each of which contains a single mutation (base change or amino acid substitution that involves one or more base changes). The latter approach is acceptable when the desired mutations are far apart. It can also be applied to closely located mutations, but the mutagenesis must be performed sequentially in multiple steps.

The QuikChange Primer Design program can design a single primer to introduce several point mutations simultaneously. For example, two amino acids that are adjacent or separated by less than four residues can be changed using one primer with all necessary base changes located in its middle portion and flanking branches that are long enough to ensure duplex stability and low energy cost.

Note that if multiple primers are required for your experiment, then the program automatically suggests using QuikChange Multi Site-Directed Mutagenesis Kit and all related free energies are calculated for 65°C. Other QuikChange kits employ the higher extension temperature of 68°C. If another QuikChange kit was selected during the initial parameter setup, but the program designed more than one primer set, then the temperature will automatically be changed to 65°C and the corresponding warning will be issued.


Mutagenesis of Untranslated DNA Sequence

Base Substitutions

The software can design primers to incorporate up to seven nucleotide substitutions with a single primer.
  1. Load sequence by pasting text or typing in the provided text area or by locating a file containing the sequence using the Browse button.
    Notes: 1) All non-"a", "c", "g", or "t" characters in the sequence will be ignored. Line breaks within the sequence are also removed during uploading.
              2) The sequence can be eather in FASTA format or plain ASCII text.
  2. Press the Upload Now button. The program will display a series of checkboxes, each corresponding to a single "sense" nucleotide from the DNA sequence.
  3. Select up to seven nucleotides that you want to change by checking the appropriate checkboxes. Then, define the new nucleotides using the drop-down lists above the checkbox area. For a single nucleotide change, use the leftmost drop-down list (Site 1). For two or more changes, use the corresponding drop-down list starting from the left. For example, if the desired mutations are 59t->c and 63a->t then check the 59t and 63a checkboxes and select "c" in the leftmost (Site 1) drop-down list and "t" in the second one (Site 2).
  4. Press Design Primers button. The program will return two tables, showing primer characteristics, sequence, and primer-template duplex schemes.


Deletions

The software can design primers to generate small deletions within a DNA sequence using a single primer.
Note: The current version of this software works well with small deletions (up to seven nucleotides). With longer deletions, the free energy of long bulge secondary structure may significantly contribute to the free energy of the primer-template duplex, and using the software to design primers for such long deletions is not recommended.
  1. Load sequence by pasting text or typing in the provided text area or by locating a file containing the sequence using the Browse button.
    Notes: 1) All non-"a", "c", "g", or "t" characters in the sequence will be ignored. Line breaks within the sequence are also removed during uploading.
              2) The sequence can be eather in FASTA format or plain ASCII text.
  2. Press the Upload Now button. The program will display a series of checkboxes, each corresponding to a single "sense" nucleotide from the DNA sequence.
  3. Check the Delete a nucleotide or a region radio button above the checkboxes.
  4. Using the checkboxes, select two nucleotides immediately flanking the deleted region as shown below:
    Sequence:        ...acgtgctagctgcacgtacgtagctacgtagcccgatcgtagc...
    Region to delete                   cacgta
    Check these bases:  ...acgtgctagctgcacgtacgtagctacgtagcccgatcgtagc... 
    Note 1) The two checked nucleotides will not be deleted
              2) If you are deleting a single nucleotide you must check the two flanking bases .
  5. Press Design Primers button. The program will open two tables with all primer characteristics, sequences, and primer-template duplex schemes.


Insertions

Note: This is only applicable for QuikChange site-directed mutagenesis kits, not for the QuikChange multi site-directed mutagenesis kit.

This software can design primers to generate small insertions into a DNA sequence using a single primer.
Note: The current software version works well with small insertions (up to 7 nucleotides). With longer insertions, the free energy of long bulge secondary structure may significantly contribute to the free energy of primer-template duplex and using the software to design primers for such long insertions is not recommended.

  1. Load sequence by pasting text or typing in the provided text area or by locating a file containing the sequence using the Browse button.
    Notes: 1) All non-"a", "c", "g", or "t" characters in the sequence will be ignored. Line breaks within the sequence are also removed during uploading.
              2) The sequence can be eather in FASTA format or plain ASCII text.
  2. Press the Upload Now button. The program will display a series of checkboxes, each corresponding to a single "sense" nucleotide from the sequence.
  3. Check the Insert between two checked nucleotides radio button, and enter the sequence to be inserted.
  4. Using the checkboxes, select the two nucleotides flanking the insertion site as shown below. The insertion will be incorporated between them:
    Check these bases     ...acgtgctagctgcgtagctacgtagcccgatcgtagc...
    Region to insert                     cacgta
    Resulting sequence:  ...acgtgctagctgcacgtacgtagctacgtagcccgatcgtagc...

    3.1 Note: two checked nucleotides must be adjacent to each other.
    Check the radio button to the left of the label: "Insert between two checked nucleotides"
  5. Press Design Primers button. . The program will open two tables with all primer characteristics, sequences, and primer-template duplex schemes.


Amino Acid Substitutions in Translated DNA

Genetic Code Table

The universal rules of DNA translation are shown in the table below. Be aware that different organisms have different codon preferences, which can be found in species-specific codon usage tables. The primers recommended by this program are optimized for the specific cloning host (Escherichia coli). Therefore, they may not comply with the codon usage preference of the organism from which the gene originated.

T C A G
T TTT Phe(F)
TTC Phe(F)
TTA Le(L)
TTG Leu(L)		 TCT Se(S)
TCC Ser(S)
TCA Ser(S)
TCG Ser(S)		 TAT Tyr(Y)
TAC Tyr(Y)
TAA Ter(*)
TAG Term(*)		 TGT Cys(C)
TGC Cys(C)
TGA Ter(*)
TGG Trp(W)
C CTT Le(L)
CTC Leu(L)
CTA Leu(L)
CTG Leu(L)		 CCT Pro(P)
CCC Pro(P)
CCA Pro(P)
CCG Pro(P)		 CAT His(H)
CAC His(H)
CAA Gln(Q)
CAG Gln(Q)		 CGT Arg(R)
CGC Arg(R)
CGA Arg(R)
CGG Arg(R)
A ATT Il(I)
ATC Ile(I)
ATA Ile(I)
ATG Met(M)		 ACT Thr(T)
ACC Thr(T)
ACA Thr(T)
ACG Thr(T)		 AAT Asn(N)
AAC Asn(N)
AAA Lys(K)
AAG Lys(K)		 AGT Ser(S)
AGC Ser(S)
AGA Arg(R)
AGG Arg(R)
G GTT Val(V)
GTC Val(V)
GTA Val(V)
GTG Val(V)		 GCT Al(A)
GCC Ala(A)
GCA Ala(A)
GCG Ala(A)		 GAT Asp(D)
GAC Asp(D)
GAA Glu(E)
GAG Glu(E)		 GGT Gly(G)
GGC Gly(G)
GGA Gly(G)
GGG Gly(G)


Amino Acid substitution and Codon Selection

This program allows you to quickly predict the most efficient base changes that result in the desired amino acid substitution. The algorithm used in this program returns all possible base substitutions to create the desired amino acid and assigns a score to each possibility. These scores are determined by the number of nucleotides that have to be changed and whether the change results in mismatches capable of base-pairing (e.g. G/T). These scores are then adjusted based on empirical data on codon bias from experiments using degenerate primers.1 The mutation with the lowest score is recommended for your mutagenesis experiment.

This software can design primers to generate up to seven amino acid substitutions with a single primer.
  1. Load sequence by pasting text or typing in the provided text area or by locating a file containing the sequence using the Browse button.
    Notes: 1) All non-"a", "c", "g", or "t" characters in the sequence will be ignored. Line breaks within the sequence are also removed during uploading.
              2) The sequence can be eather in FASTA format or plain ASCII text.
  2. If you don't specify the optional translation region within the DNA sequence, then the only F1 translation will be performed, otherwise, only frame 1 (F1) of the specified region will be translated. Press Translate button. This will display a number of checkboxes, each of which will correspond to a single amino acid residue from the translated DNA sequence.
  3. Select up to seven amino acids that you want to change. Then define the target residues using the drop-down lists below the checkbox area. For a single amino acid change use the leftmost drop-down list; for two or more changes use corresponding drop-down lists starting from the left. For example if the desired mutations are 25A->R and 27T->M then you have to check 25A and 27T checkboxes, and select "R(arg)" in the leftmost drop-down list and "M(met)" in the second one.
  4. Press Design Primers button. The program will open two tables with all primer characteristics, sequences, and primer-template duplex schemes.
Deletion of amino acids can be performed by deleting the codon from the DNA sequence using the protocol for deleting nucleotides. This program does not specifically assist with insertion of amino acids due to possible genetic code ambiguity. Therefore, we recommend designing primers for amino acid insertion by working with the untranslated DNA sequence and using unambiguous codons for the needed insert.

Reference:

1.Novoradovsky, A.,et al. (2005). Computational Principles of Primer Design for Site Directed Mutagenesis. Technical Proceedings of 2005 NSTI Nanotechnology Conference and Trade Show, Anaheim, 2005, pp. 532-535.



DISCLAIMER

This software is designed to provide free service for site-directed mutagenesis. By using this software, such users accept and agree to the following:

THIS SOFTWARE IS PROVIDED "AS IS." ALL EXPRESS OR IMPLIED CONDITIONS, REPRESENTATIONS, AND WARRANTIES INCLUDING, WITHOUT LIMITATION, ANY IMPLIED WARRANTY OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NONINFRINGEMENT, OR ANY IMPLIED WARRANTY, RIGHT OR OBLIGATION ARISING FROM A COURSE OF DEALING, USAGE, OR TRADE PRACTICE, ARE HEREBY EXCLUDED AND EXPRESSLY DISCLAIMED TO THE EXTENT ALLOWED BY APPLICABLE LAW. THE UNIFORM COMPUTER INFORMATION TRANSACTIONS ACT, WHEREVER ENACTED, SHALL NOT APPLY TO THE ACQUISITION OR USE OF THIS SOFTWARE.

IN NO EVENT WILL STRATAGENE BE LIABLE FOR ANY LOSS, EXPENSE OR DAMAGE, OF ANY TYPE OR NATURE, INCLUDING WITHOUT LIMITATION, ANY LOST REVENUE, PROFIT, OR DATA, OR FOR SPECIAL, INDIRECT, CONSEQUENTIAL, OR INCIDENTAL DAMAGES HOWEVER CAUSED AND REGARDLESS OF THE THEORY OF LIABILITY ARISING OUT OF THE USE OF OR INABILITY TO USE THE SOFTWARE EVEN IF STRATAGENE HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. Some states do not allow limitation or exclusion of liability for consequential or incidental damages.

THE RISK OF ANY AND ALL LOSS, DAMAGE, OR UNSATISFACTORY PERFORMANCE OF THIS SOFTWARE RESTS WITH THE USER. TO THE EXTENT PERMITTED BY LAW, STRATAGENE MAKES NO REPRESENTATION OR WARRANTY, EITHER EXPRESSLY OR IMPLICITLY, REGARDING THE APPROPRIATENESS OF THE USE, OUTPUT, OR RESULTS OF THE USE OF THIS SOFTWARE IN TERMS OF ITS CORRECTNESS, ACCURACY, RELIABILITY, OR OTHERWISE. STRATAGENE SHALL HAVE NO OBLIGATION TO CORRECT ERRORS, MAKE CHANGES, SUPPORT THIS SOFTWARE, DISTRIBUTE UPDATES, OR PROVIDE NOTIFICATION OF ANY ERROR OR DEFECT, KNOWN OR UNKNOWN. IF YOU RELY UPON THIS SOFTWARE, YOU DO SO AT YOUR OWN RISK, AND YOU ASSUME ALL RESPONSIBILITY FOR THE RESULTS.