Transcript Primer Designer for Site-Directed Mutagenesis Alexey

Primer Designer for Site-Directed Mutagenesis
Alexey Novoradovsky1, Vivian Zhang, Madhushree Ghosh2,
Holly Hogrefe2, William Detrich2, Joseph A. Sorge2, Terry Gaasterland1.
DISCUSSION
INTRODUCTION
QuikChange® Primer Designer is a part of the Stratagene LabTools website:
http://labtools.stratagene.com. This software designs efficient primers for site-directed
mutagenesis by calculating free energy of the mismatched primer-template duplex and
comparing it to the energy of perfect duplex. The result of such comparison is interpreted
as an “energy cost” of a mismatch. Preference in primer design strategy is directed
toward minimizing of the energy cost, which also results in fewer nucleotide substitutions
or result in mismatches capable of base-pairing (e.g. G/T).
In addition to energy conservation rules, we analyzed data obtained with degenerate
primers and incorporated several empirical rules regarding optimal codon replacement,
which were also included in this software.
METHODS
Codon Selection:
If multiple codon choices are possible then the preference is made in favour of fewer base
changes. For example, if threonine encoded by ACG is to be mutated to arginine then the
desired change can be performed by six different ways: AGA, AGG, CGA, CGC, CGG, or
CGT. However the program would recommend AGG as a target, because it only involves
a single base change.
Further improvements of the codon selection were introduced by the results of SDM
experiments with the degenerate primers. In these experiments, primers contained a
single degenerated codon “NNK” (which includes 32 possible triplets, encoding for 19
amino acids: ACDEFGHILMNPQRSTVWY). The numbers of resulted mutant DNA
sequences were counted among 607 mutants, and the codon biases were calculated as a
deviation of the observed number of resulting codons (NO) from expected 1/32 of the total
number of analyzed mutants (NE). The codon biases ranged from -12 to +23 (Table 1).
1. The Rockefeller University, New York, NY 10021
2. Stratagene Cloning Systems, La Jolla, CA 92037
ABSTRACT
Engineering mutations within cloned DNA fragments involves the design of primertemplate duplexes containing the target mutation. These primers have single or multiple
base mismatches, or in the case of a deletion or an insertion, single-stranded DNA loops.
Such DNA duplexes have a higher free energy than a mismatch-free perfect duplex. Our
program suggests the most energy-saving, and thus the most effective, base
substitutions to generate single or multiple amino acid changes, frame-shifts, deletions,
or insertions within the target DNA molecules. Preference in codon replacement is given
to the codon changes that involve fewer nucleotide substitutions and are more
energetically favorable. The energy “cost” is calculated as a difference between the
summary stacking and nearest-neighbor energies of the perfect primer-template duplex
compared to the duplex with mismatches. In addition to free energy conservation
principles, the software incorporates several empirical rules for nucleotide changes,
which were established through mutagenesis experiments using degenerate primers.
The program is written in PHP and is accessible as a free web service in the Stratagene
web site: http://labtools.stratagene.com
NO-NE Codon
-12.0
CGT
-12.0
GTT
-9.0
AGT
-9.0
TTT
-8.0
GGT
-7.0
TAT
-7.0
TCG
-6.0
TGG
NO-NE
-6.0
-6.0
-5.0
-4.0
-4.0
-3.0
-1.0
-1.0
Codon
GGG
CAG
GAT
ATG
GTG
CAT
AAT
CGG
NO-NE
0.0
0.5
1.0
2.0
2.0
3.0
3.0
4.0
Codon
GCT
GCG
ATT
ACG
TAG
AGG
AAG
GAG
NO-NE
4.0
4.0
5.0
6.0
11.0
13.0
22.0
23.0
Further information about the Primer Designer for Site-Directed Mutagenesis can be
obtained through the help hyperlinks available to the registered users. Registration for
this service, as well as for other free Stratagene LabTools services, can be obtained at
http://labtools.stratagene.com.
Primer Designer for Site-Directed Mutagenesis service was launched in July 2003, and
during the last eight months the user database includes about 7,000 users.
CONCLUSIONS
1. Primer design for site-directed mutagenesis was addressed through integration of
primer-template duplex energy calculation with the data on codon biases established
through SDM experiments with degenerate primers.
2. PHP-enabled web interface has been created to provide user access to the databasedriven server-side applications.
REFERENCES
Table 1. Codon biases revealed by the SDM experiments with degenerate primers.
Codon
TCT
CTT
TGT
CCT
CCG
CTG
ACT
TTG
The example in Figure 1 demonstrates one of the four available primer design routines,
single amino acid change. Other applications, that are not illustrated here, include: 1)
optimization of codon selection, described in Methods; 2) design of single or multiple
primers with up to five single-nucleotide changes; 3) design of multiple mutagenesis
primers for simultaneous introduction of up to five amino acid changes in one
experiment, implemented by Stratagene’s QuikChange® Multi kit; and 4) design of
primers to generate deletions or insertions in the target DNA sequence.
1. Nielsen, D.A., Novoradovsky, A., Goldman, D. SSCP primer design based on
single-strand DNA structure predicted by a DNA folding program. Nucleic Acid
Research, 1995, 23: 2287-2291.
2. SantaLucia, J. Jr., Allawi, H.T.,
Seneviratne, P.A. Improved nearestneighbor parameters for predicting DNA
duplex stability. Biochemistry, 1996, 35:
3555-3562.
3. http://labtools.stratagene.com
1. Select program.
2. Using available options, enter
DNA sequence, its format and
translation range.
Codons with the highest positive biases contain significantly higher proportions of
dinucleotides AG, AT, AA, TA, AC, and GA, while dinucleotides CT, TG, TT, CC, and GT
were underrepresented among observed mutants. These observations are also
considered by the codon selection program.
Figure 1.
Primer Design:
Free energy of a primer-template duplex was calculated as a sum of stacking energies of
nearest neighbours. Mismatches in a duplex were treated as the internal loops or bulges,
flanked by perfect duplex branches. The calculation principles derived from the nearestneighbour energy calculation of nucleic acid duplex (Nielsen et al, 1995, SantaLucia et
al.,1996). They are based on summarizing the nearest dinucleotide-duplex energies,
where the members of the “tetrads” can be either parts of perfectly matched branch
duplexes or the internal loops/bulges or parts of terminal “dangling ends”.
Web interface:
Primer design process is piped through the submission of HTML forms with generating
and passing variables via PHP scripts. Registered SDM primer designer users are
validated through the PostgreSQL user database containing usernames and encrypted
passwords. All possible matched or unmatched nucleotide “tetrads” at temperatures from
37oC to 85oC are stored in another PostgreSQL database, which is accessed through
PHP postgres functions. All the calculations are performed through PHP scripts.
The example of using QuikChange® Primer Designer to design a single SDM
primer for mutating Methionine-77 to Arginine. 1, program selection can be one
using the left-side menubar or using the hyperlinks in the page body; 2, DNA
sequences can be entered as flat-text FASTA or GenBank-formatted files either
using the file control or directly by copying-pasting to the provided textarea. If
amino acid changes are to be performed then the translated sequence can be
displayed at the next step either completely or within specified translation range.
4b. (Optional): view position of
the designed primer within DNA
template.
3. Define amino acid change
and annealing temperature.
Otherwise, the untranslated DNA sequence will be displayed; 3, amino acid change must be specified by checking
the ”radio” button in the gray area and selecting the target amino acid (or termination codon) from the drop-down
menu; 4a, recommended ”sense” and ”antisense” primers with melting temperature close to 80oC are shown in the
table. The associated free energy of the mismatched primer-template duplexes, and the ”energy costs” associated
with the suggested are also displayed. Note: the Designer automatically suggests the most ”energy-cost-effective
codon AGG as requiring the only base change, and containing ”good” dinucleotide AG (see METHODS). The
hyperlink to ”template sequence” at the top of the page can be used to view the position of the designed primer
within DNA template.
4a. View designed ”sense” and ”antisense” SDM primers and
their characteristics.