Abstract
Site-directed mutagenesis is a powerful tool that has enabled molecular biologists to perform functional analysis of altered nucleic acids and proteins3. Newer PCR-based mutagenesis techniques have reduced the process of mutagenesis to as little as one day2. While each technique has its advantages, both require a strategy to isolate the desired clone from a population that contains mutagenized and wild type genes. In this report, we describe a World Wide Web-based computer program that facilitates the design of mutagenic primers such that successfully mutagenized clones can be identified by the presence or absence of a unique restriction site.
Introduction
Many site directed mutagenesis protocols call for the use of two oligonucleotides5. The mutagenic oligonucleotide introduces the desired mutation and a selection oligonucleotide increases the fraction of successfully mutagenized clones and facilitates their subsequent identification. The selection oligonucleotide does improve the fraction of mutagenized clones but it is not 100% effective3. Clones to which the selection oligonucleotide but not the mutagenic oligonucleotide has annealed are only identified with subsequent DNA sequencing.
If the mutagenic primer introduces or deletes a restriction site, then it can aid in the subsequent identification of mutagenized clones. In effect, the mutagenic primer can be carefully designed to also be a selective primer. The Primer Generator automates the selection of a mutagenic oligonucleotide that meets these criteria. This technique, of carefully selecting a mutagenic oligonucleotide to facilitate subsequent mutant identification, is a useful and cost effective method for optimizing the procedure of site directed mutagenesis.
Materials and Methods
Software
The following software is used: Perl10 with a CGI module9 and NCSA HTTPd server6.
Algorithm
An outline of the program can be seen in figure 1. Initially, the investigator inputs the nucleotide sequence encoding the current protein fragment and the protein sequence desired following mutagenesis. The latter sequence is reverse translated using the redundancy of the genetic code to produce a number of DNA sequences that could potentially encode the given protein sequence.
Following reverse translation, the sequences are interrogated for restriction enzyme sites using data from The Restriction Enzyme Database (REBASE) at http://rebase.neb.com/rebase/rebase.html. The results of these searches are independently compiled for the current and all possible target nucleotide sequences. Potential restriction sites in the existing sequence are subtracted from restriction sites in the mutagenized sequence thereby generating a list of restriction sites unique to the mutagenized product. Similarly, restriction sites in the mutagenized sequence are subtracted from restriction sites in the existing sequence which identifies sites that were deleted by the mutagenesis procedure.
From these operations, a list of unique restriction sites in the mutagenized sequence and the oligonucleotide probes needed to generate them is compiled. In addition, restriction sites that are deleted from the existing sequence through the mutagenesis procedure are also listed. The mutagenic primer output is ordered by increasing number of base pairs that differ from the original sequence. These base pairs are boldfaced. A useful feature of the pPrimer gGenerator is the ability to limit the output by the maximal number of substitutions that will be tolerated in the mutagenic primer. It can be set for 1 through 10 or for no limit. This feature facilitates selection of a mutagenic primer that necessitates minimal alteration of the existing sequence. To decrease computation time, especially for longer sequences, it is also possible to limit the number of restriction endonucleases to only the most commonly used enzymes. These enzymes are likely to be readily available in most laboratories.
Introducing or deleting a unique restriction site may necessitate mutagenizing more than the minimal number of residues required to elicit the desired change in primary sequence. The mutagenesis procedure is sufficiently robust however to facilitate changing more than one base pair. A mutagenic oligonucleotide may also be chosen such that it introduces a unique restriction site in the mutagenized sequence, deletes a specific restriction site from the existing sequence or produces some combination of the two. A number of clones from the mutagenesis procedure can then be screened in a short amount of time with a restriction digest prior to being sequenced.
The restriction enzymes' names in the output are linked to the corresponding entries in the REBASE database which provides the user with additional information about the enzyme. Included among this information is enzyme type, microorganism from which it was isolated, the prototype of the enzyme (the isoschizomer that was discovered first), the recognition sequence, the sites at which the DNA strand is cleaved, commercial sources of the enzyme and references pertaining to the discovery and characterization of the enzyme.
Results
To demonstrate the effectiveness of this algorithm, we asked the program to develop primers that mutagenize a critical serine residue in the Rapamycin And FKBP12 Target protein 1 (RAFT1) to arginine. When administered to cells, rapamycin forms a complex with the immunophilin FKBP12. This complex subsequently interacts with RAFT18/FRAP1. This FKBP12-rapamycin-RAFT1 interaction is dependent on the presence of a serine residue at position 2035 in RAFT1/FRAP1. RAFT1/FRAP mutants that do not complex with FKBP12 in the presence of rapamycin are proving instrumental in elucidating the signaling capacity of this protein. The output of the program is summarized in figure 2.
The output in figure 2 is only a representative selection of the entire output of the program which included 15 different mutagenic oligonucleotides. The mutagenic oligonucleotides (limited to the maximum of three substitutions compared to the original sequence) are capable of introducing 14 unique restriction sites, deleting the 5 existing sites and allowing the user to select from combinations thereof. Alternatively, with settings changed to limit restriction endonucleases used in analysis only to the most common enzymes, and the maximum number of substitutions raised to four, we are still able to introduce six new and remove two old restriction sites (data not shown).
Discussion
The Primer Generator has a number of uses. For example, an approach that has been used to define the binding specificities of modular protein-protein interaction domains such as the phosphotyrosine directed SH3 domain is to independently vary the amino acid residue at a single position to the other 19 amino acids7. This method has been successful in defining the binding characteristic of a single position (hydrophobic, basic, etc.) in the target peptide. By using the Primer Generator, one could order a single mutagenic primer that contains 3 N's in place of the codon that is to be mutated as well as an N’s in the wobble position of the adjacent invariant codons (over a thousand different sequences). A single mutagenesis reaction would be performed, and a few well chosen restriction digests would rapidly identify the desired mutants while preventing the sequencing of redundant mutants.
In addition to its use for mutagenesis, the pPrimer gGenerator can also be of use in clinical settings. A number of genetic diseases such as cystic fibrosis are characterized by a specific mutation that accounts for greater than 70% of all cases101. If one wished to screen PCR products generated by primers flanking the region most commonly mutated, they need only input the wild type and mutant sequences into the program to find restriction enzymes that can be used to distinguish between the two.
Computers are assuming an ever increasing role in molecular biology. The repertoire of computer-based tools and utilities available to investigators continues to grow. Here we report an algorithm that allows one to choose a mutagenic oligonucleotide such that successfully mutated DNA can readily be identified via a restriction digest. The flexible, user friendly interface offers the user extensive control of the program and its output. The hot links provide the user with additional information about the enzyme chosen. The availability of site-specific, hybrid restriction endonucleases is expected to increase the number of enzymes available for use by this program4. A carefully chosen mutagenic oligonucleotide can save time and money by reducing the number of primers used and clones sequenced. The Primer Generator can be accessed at http://www.med.jhu.edu/medcenter/primer/primer.cgi
Acknowledgments
The authors wish to thank Dr. Robert Cottingham and Dr. Peter Li for constructive feedback and technical comments. We are also indebted to the Division of Biomedical Information Sciences of the Johns Hopkins University School of Medicine for allotting the project free space on the JHMI Affiliates Server.
References
