Primer Design Software Research Articles

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).

Set of novel tools for PCR primer design.

We have developed a new package of computer programs and algorithms for different PCR applications, including allele-specific PCR, multiplex PCR, and long PCR. The package is included in the upcoming VectorNTI suite software and attempts to incorporate most of the current knowledge about PCR primer design. A wide range of primer characteristics is available for user manipulation to provide improved efficiency and increased flexibility of primer design. To accelerate the primer calculations, we have optimized algorithms using recent advances in computer science such as dynamic trees and lazy evaluation. Proper structural organization of input parameters provides further program acceleration. New Vector NTI primer design software allows calculations of primer pairs for long PCR amplification of 120-kb genomic DNA in 5 min under most stringent input parameters and clustering 435 primer pairs for multiplex PCR within 30 min on a standard Pentium III PC. Our program allows the user to take advantage of molecule annotation by applying different kinds of filtering features during PCR primer design.

How Quantitative is Quantitative PCR with Respect to Cell Counts?

Quantitative diagnostic PCR systems based upon rDNA targeted primer and probe combinations were developed for the detection of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, enterococci, Staphylococcus aureus, and Staphylococcus epidermidis. Primers and probes were designed in silico using the ARB software package (TU Munich) in combination with Primer Design software of PE Applied Biosystems. Purified genomic DNA or bacterial cells of target and reference organisms were used for the evaluation of the PCR assays applying the TaqMan technique on an ABI PRISM TM 7700 Sequence Detection System (PE Applied Biosystems). Sensitive, reliable and reproducible quantification of target rDNA could be achieved applying primer-probe combinations that mediate in vitro amplification of DNA fragments smaller than 100 base pairs. Large amounts of non target DNA (1 mg per sample) remarkably affected the quantification potential of the approach resulting in an underestimation of the amounts of target DNA. One of the principal goals was to use quantitative PCR to study the correlation of gene and cell numbers depending on the growth behavior of target organisms and to explore the potential to estimate cell numbers from target DNA quantification. A clear correlation of rDNA quantification and bacterial growth was observed, however, cell numbers cannot directly be estimated from quantitative PCR data, given that the cellular genome content varies with the growth phase of the organisms. In the case of Escherichia coli the cell numbers which could be assigned to a certain number of rDNA targets varied reasonably depending upon the growth phase of batch cultures.

General concepts for PCR primer design.

1Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; ZDepartment of Molecular Biology, Washington University, St. Louis, Missouri 63110; 3Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 PCR is a technology born of the modern molecular biology era. The enzyme used for PCR, Taq DNA polymerase, supplied with the 10x buffer, is purchased as a cloned product, and the nucleoside triphosphates are ultrapure, buffered, and available at a convenient concentration. Yet, with all of these commercially available starting materials, PCR still fails, particularly for the novice. Assuming that all of the reagents have been added in the proper concentrations, two critical PCR components are left to the researcher. The first is the nucleic acid template, which should be of sufficient and contain no inhibitors of Taq DNA polymerase (although when it comes to template purity, PCR is more permissive than many other molecular biology techniques). The second is the selection of the oligonucleotide primers. This process is often critical for the overall success of a PCR experiment, for without a functional primer set, there will be no PCR product. Although the selection of a single primer set may be trivial, the construction of primer sets for applications such as multiplex or nested PCR becomes more challenging. The manual selection of optimal PCR oligonucleotide primer sets can be quite tedious and thus lends itself very naturally to computer analysis. The primary factors that affect the function of the ol igonucleotides-their melting temperatures as well as possible homology among primers--are welldefined and straightforward tasks that are easily encoded in computer software. Once the computer has provided a small number of candidate primer sets, the task of selection can be (and still is) performed manually. In this approach, the researcher is taking advantage of the raw speed of computer calculations, trying all possible permutations of a primer's placement, length, and relation to the other primers that meet conditions specified by the user. From the thousands of combinations tested by the computer, a software program can present just those that are suitable for the needs of the experiment. Thus, the overall quality (as defined by the user in program parameters) of the primers selected is almost guaranteed to be better than the handful chosen and hand-tested by the research without computer assistance. As with any tool, understanding its function will make the end product more useful. A wide range of programs have been written to perform primer selection, varying significantly in selection criteria, comprehensiveness, interactive design, and user-friendliness. (1-1~ There are also commercial ly available specialty primer design software programs that offer enhanced user interfaces, additional features, and updated selection criteria, (1'2) as well as primer design options that have been added to larger, more general software packages. Although most people would agree that application of analytic computer software to a well-defined problem is a smart thing to do, not all researchers are convinced that PCR primer selection is a nontrivial task, or that the selection rules that make a primer amplify efficiently are even well defined. Even though many of the rules discussed have been fine-tuned by collective empirical wisdom, most are based on firm theoretical ground, if not c o m m o n sense. The purpose of this chapter is to explain basic rules of oligonucleotide primer design. With this understanding of primer selection criteria, the information deduced by primer design software can be rationally interpreted and manipulated to fit your experimental needs.