Online primer design tool
ONLINE PRIMER DESIGN TOOL SOFTWARE
Currently, this can be achieved only by transporting the data between several software applications, each of which is specialized for use at a different stage of the process. Assay development relies on the availability of genome databases containing the identity and location of relevant SNPs for input into primer design software applications. The development of Pyrosequencing assays can be accomplished by exploitation of the sequence databases derived from the human and model organism genome projects and can be combined with primer design algorithms when large batches of SNPs are to be analyzed. Thus, none of the existing software applications available via the Internet are capable of designing the complete set of primers required for Pyrosequence-based typing (PSBT) during SNP-scanning projects while also allowing for the simultaneous development of assays for multiple SNPs. Moreover, applications currently available for developing Pyrosequencing primers do not also allow for the design of primers for PCR. Publicly and commercially available software applications have been developed for PCR primer design, but these have not taken into account the added constraints that are essential when optimizing Pyrosequencing reagents ( 9, 10).
![online primer design tool online primer design tool](https://sfvideo.blob.core.windows.net/sitefinity/images/default-source/default-album/decoded-temp-image-storage/doart11-wt-fig1-screen_choosedesign.jpg)
The design of oligonucleotide primer sets for use during PCR and Pyrosequencing, a necessary step in the process of Pyrosequence-based mapping of genetic disease loci, while not individually cumbersome, is time-consuming to develop and test when large numbers of SNP-containing sites are to be examined. The Pyrosequencing reaction is quantitative in that increased light intensity is produced upon incorporation of multiple nucleotides. The incorporation of a particular nucleotide is displayed graphically in the form of a pyrogram of nucleotide dispensation event versus the intensity of emitted light. The detection of nucleotide sequence is performed by way of a chain of enzymatic reactions involving the activities of DNA polymerase, apyrase, ATP sulfurylase, as well as luciferase ( 1, 5). Pyrosequencing is performed by the addition of dNTPs individually, in a predefined dispensation order, so that the nascent nucleotide chain is extended one nucleotide residue per dispensation event. The method is suited for use during the analysis of single nucleotide polymorphisms (SNPs) and the detection of genomic insertion and deletion polymorphisms because of its ability to provide high-quality sequencing for short stretches of DNA and to accurately resolve heterozygous nucleotides by enabling out-of-phase sequencing ( 8). Sequencing of lengths between 50 and 150 nucleotides has been reported during HLA genotyping as well as during the analysis of PCR-amplified cloned DNA ( 4–7). Pyrosequencing® has been developed to allow for accurate sequencing of short stretches of DNA, initially for the analysis of expressed sequence tags (ESTs References ( 1–3). Of the SOP 3-designed primer sets that were tested, a large majority of the primer sets have successfully produced PCR products and Pyrosequencing data. The method has been tested in the development of Pyrosequencing assays for determining SNPs and for deletion/insertion polymorphisms in the human genome. Theoretical pyrograms for each allele are calculated and presented graphically. SOP 3 presents for each entry a set of forward and biotinylated reverse PCR primers as well as a sequencing primer for use during the analysis of SNPs by Pyrosequencing. The location of an individual polymorphism, such as an intron, exon, or 5′ or 3′ untranslated region is indicated, as are whether nucleotide changes in an exon are associated with a change in an amino acid sequence. Output can be parsed by gene name, SNP reference number, heterozygosity value, location, chromosome, or function. The application accepts as input gene name, SNP reference sequence number, or chromosomal nucleotide location.
![online primer design tool online primer design tool](https://www.selectscience.net/images/articles/4417_IDT-Primerquest.jpg)
Accessible via the Internet, the application is optimized for developing the PCR and sequencing primers that are necessary for Pyrosequencing®. SOP 3 is a web-based software tool for designing oligonucleotide primers for use in the analysis of single nucleotide polymorphisms (SNPs).