polymerase chain reaction ( pcr ) is a commonly used laboratory procedure nowadays for a variety of tasks , such as dna cloning , sequence determination and snp detection .
consequently , numerous primers need to be designed for dna amplification in order to produce enough dna . for dna sequencing , to design primers for the promoter and exon regions of a gene , one needs to retrieve the required sequence information for each single exon and promoter , including their corresponding flanking sequences , convert them to the correct format , switch to a primer design application like primer 3 ( 1 ) , import the sequence information and adjust parameter settings if necessary before submitting the request . while this is tolerable for primer design for a gene that has a single exon , a human , mouse or rat gene can easily have more than 10 exons , and each of the above steps may thus need to be repeated 10 times or more .
furthermore , if an exon is too long to sequence properly in one run , several primers have to be designed to map overlapping sections of the exon .
the whole process requires many manual steps for repeated window opening , browsing , application switching , copying and pasting , typing and so on , which can be tedious , time - consuming and error prone .
in fact , for a gene with 10 exons , the process can easily exceed 300 steps . to improve the situation , we have developed primerz to replace almost all of these manual steps so that users can easily complete a primer design task using just a few clicks for a gene or batched human snps .
more than 2000 primers have been designed with primerz at our institute since 2004 and the success rate is over 70% .
there are a multitude of user - definable options available including product size , maximum exon length , excluded regions of the query sequence , gc - content and maximum allowable local alignment score . simply by submitting a candidate gene name ,
a snp i d or up to 100 batched snp ids , in most cases all the primer sequences should be returned in a minute or two . the generated information , including gene transcript graph , primer data from primer3 , and direct links to ucsc in - silico pcr ( 3,4 ) for pcr product prediction , to ncbi blast and to ensembl source data , is integrated and displayed on a single page for convenient viewing .
additionally , all the primer data can be exported in csv format for further processing .
primerz takes advantage of the well - developed public - domain database ensembl , through its api ( application programming interface ) .
when a gene query is received , primerz will access the ensembl database through ensj api ( 5 ) to retrieve promoter and exon information . by default but with an adjustable setting ,
primerz retrieves one region of 1440  bp upstream from the start of 5-utr which it treats as the promoter region .
the promoter region is thereafter divided into four 360  bp non - overlapping segments plus their respective flanking sequences .
all exons are directly flanked with 240  bp sequences , except when an exon of the gene is > 360  bp , when the sequence will be split into segments of 360  bp for a better quality sequencing result .
for example , an exon of 1000  bp will produce two 360  bp segments and one 280  bp segment . the above sequence information , plus parametric settings packaged by primerz are then fed into primer3 for primer design .
all returned primer results , together with a transcript graph , are integrated into a one - page report for final output .
the self - explanatory workflow of the whole primerz system is shown in figure 1 . 

the software development environment included the following software : 
 java , jdk : j2sdk1.4.2_06 , server vmstruts framework 1.2red hat enterprise linux academic editionmysql 4.1tomcat 5.0 web serverensembl java api java , jdk : j2sdk1.4.2_06 , server vm red hat enterprise linux academic edition tomcat 5.0 web server the api provided by ensembl is used for gene information retrieval .
java language is used to pipeline gene data into primer 3 and merge the returned results .
primerz is an easy - to - use tool to design primers for genes and snps , using only a few simple steps to design the wanted primers .
it currently provides gene primer design for all ensembl species while snp primer design is currently only available for human snps . for gene primer design ,
there are some essential parameters required for optimal design of primers , such as maximum exon length , exon flanking region , product size range and excluded region .
the  excluded region value allows the program to bypass regions with low sequence quality or containing repetitive elements such as alus or lines for primer design . for snp primer design , a snp rsid or an affy_probid(6 ) as well as a batch file containing mixed
i d types can be accepted as input , where the maximum number of snps per batch is limited to 100 .
the result page comprises three major parts : initial input data and parameters , the gene transcript diagram ( only available for gene primer design ) and designed primer information .
the first part lists all the input data and parameters plus ensembl database version , for ease of reference .
the second part shows each promoter fragment and exon of a transcript diagram , and links to their accompanying original primer3 output . finally , the third section presents tabulated primer information and incorporates executable links to ncbi blast and ucsc in - silico pcr to check specificity and product prediction .
ensembl link button offers the ensembl exon report of the transcript so that a user can trace all primers to their original sequence information .
a csv format text file of the results can be downloaded at this results page .
in a manual operation benchmarking test , it took a person very familiar with all processes about 1260  s and 380 discrete steps to design the primers of hadhsc ( l-3-hydroxyacyl - coenzyme a dehydrogenase , short chain ) , our benchmark gene with 10 exons .
with automated primerz , the same operation took only 16  s and three steps .
this comparison clearly demonstrates the superior efficiency and ease - of - use of primerz for gene primer design .
a benchmark test on batched snps showed a similar dramatic reduction in workload from 90 discrete steps to two steps , from 840 to 49  s , and from 40 result pages to a single page .
primerz has been designed to obtain reliable primers for pcr experiments and to allow standardized , automated primer design for batch operation .
users can access the ucsc in - silico pcr directly from the result page to verify their primers to achieve higher accuracy and lower cost .
primerz also allows users to modify the conditions of primer design , including the maximum exon size , the flanking region of the target sequence , the exclusion region and the maximum allowed polya and ca - repeats in the pcr products .
in addition , primerz will offer primer design from ncbi transcripts in the near future , which should be of great interest to those users who use ncbi data to design primers .
the results from ncbi and ensembl will be shown in the same page . following the release of primer3 web interface in november 2006 , we are installing and testing a local copy of primer3 web to alleviate the burden on the original primer3 website and the restriction on the number of snps allowed .
primer z is a simple - to - use program that greatly facilitates and enhances the traditionally time - consuming task of accurate primer design for pcr , and should be an excellent additional tool for the modern molecular biologist .
 primerz ( http://genepipe.ngc.sinica.edu.tw/primerz/ ) is a web application dedicated primarily to primer design for genes and human snps . 
 primerz accepts genes by gene name or ensembl accession code , and snps by dbsnp rs or affy_probe ids . 
 the promoter and exon sequence information of all gene transcripts fetched from the ensembl database ( http://www.ensembl.org ) are processed before being passed on to primer3 ( http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi ) for individual primer design . 
 all results returned from primer 3 are organized and integrated in a specially designed web page for easy browsing . besides the web page presentation , csv text file export is also provided for enhanced user convenience.primerz automates highly standard but tedious gene primer design to improve the success rate of pcr experiments . 
 more than 2000 primers have been designed with primerz at our institute since 2004 and the success rate is over 70% . 
 the addition of several new features has made primerz even more useful to the research community in facilitating primer design for promoters , exons and snps . 