ExPrimer: to design primers from exon—exon junctions

Abstract

Summary: ExPrimer is a web-based computer program to design primers mainly from a specified exon—exon junction (E-E-jn) of a gene of interest. The tool suggests the optimum primer-pair(s) of which the right (reverse) primer represents a particular E-E-jn of the mRNA. The ‘product length’ decides the location of the left primer. The results also include all other primer pairs considered and their ‘scores’. ExPrimer can use the NCBI BLASTn program for sequence specificity of primers. The tool is useful in many areas of molecular biology research that involve hybridization of short sequences with mRNA or cDNA.

Availability:  http://exprimer.ibab.ac.in/exprimer_html/exprimer.html

Contact:  [email protected]

Identification of appropriate short nucleotide sequences (i.e. ‘designing the primers’) is crucial for several techniques that involve DNA/RNA hybridization. Various primer-parameters such as GC content, melting temperature (T_m), self annealing, end annealing etc. (Sambrook and Russel, 2001 and the references cited therein) have to be analyzed while designing primers. Many ‘general’ primer designing computer tools can quickly perform such an analysis to suggest the optimum primer pair within a specified region of nucleotide sequence.

The location for primer selection becomes crucial for certain types of experiments. It is often necessary to choose the primers from the exon—exon junctions (E-E-jns), the regions of mRNA which include nucleotide sequences from the 5’ border of one exon and 3’ border of the neighboring exon. Selecting at least one primer that represents one such E-E-jn of a particular transcript is important for expression profiling of alternatively spliced variants. The E-E-jn specific oligonucleotides could also be used as probes in microarray experiments addressing alternative splicing and the follow up confirmations by reverse transcription—polymerase chain reaction (RT—PCR) (e.g. Johnson et al., 2003). This type of ‘junction primers’ could be used to overcome the problem of genomic DNA in mRNA samples used in RT—PCR reactions. Thus, there are several research questions and technical situations in molecular biology which require E-E-jn specific oligonucleotides from the transcripts of genes of interest. Selecting an appropriate sequence from E-E-jns manually is a tedious and time-consuming process. Using currently available ‘general’ primer designing tools or software will reduce these problems to some extent only because selection of primer source (E-E jn) sequences still needs to be done manually. Hence, there is a need for a program that could help molecular biologists to obtain the best primers from the specific E-E-jn in a particular transcript of the gene of interest. Here we present such an online tool which can also help in the sequence analysis of primers by interfacing with the NCBI BLASTn program.

When this manuscript was being written, a non-peer reviewed article (Wrobel et al., 2004) reported a similar online software, ‘AutoPrime’. This tool uses the Primer3 program to generate primers from E-E-jns. Though ExPrimer has many objectives similar to Autoprime, there are significant differences in the approach and results. For example, Autoprime uses the Ensemble database to retrieve sequences, while ExPrimer relies on the user-defined GenBank file. Unlike the common trend to use the mis-priming library of repetitive sequences, ExPrimer uses BLASTn to evaluate mis-hybridization of the primer sequences to the genome/transcriptome. Thus, the primers’ sequence specificity is determined by comparing it with most of the known sequences. ExPrimer also provides details of self and pair annealing alignments.

ExPrimer suggests best possible primer-pair(s) by the reference point approximation method (Kampke et al., 2001). A vector of scores for different primer-parameters and its distance from a ‘reference vector’ are calculated. The reference vector is composed of the user defined parameters (GC content, T_m etc.) and a few internally set values (such as self annealing and pair annealing scores). There is a penalty assigned for every significant difference between the reference vector and the calculated primer-parameters of each primer-pair. The primer pair(s) with the lowest final score (total of all penalties) is/are suggested as the best (Kampke et al., 2001).

Input to ExPrimer is the GenBank file for the genomic contig of interest. ExPrimer has a link for easy access and downloading of the GenBank file. This file provides the start and end positions of the exons of the gene of interest. This information could be utilized to decide the approximate position of the desired primers. Based on exon locations, one could choose the appropriate E-E-jn, from where the right primer is selected, and the product length, which decides the location of the left primer (Fig. 1). The GenBank file also contains ‘transcript ID’ for every transcript. The user needs to note the ID of the specific transcript and enter it in ExPrimer’s initial page. Using this ID, ExPrimer selects the source sequence from the GenBank file to generate the primers (Fig. 1).

Currently, three versions of ExPrimer are available on the web: BLAST single step, BLAST two step and non-BLAST. In the BLAST versions, the primer source sequences are automatically submitted to the NCBI BLASTn program in a predefined query format. The user needs to save the BLAST output as a text file in the BLAST two-step version which in turn is the input for ‘primer generation’, the second step; primer designing parameters can be set on the primer generation page to generate the final output. In the BLAST one-step version, the primer generation phase is incorporated in the first page and the user does not get to see the BLAST result details. The BLAST scores in both the versions, however, appear on the ‘results’ page. The accession numbers and corresponding NCBI links for the sequences with the highest homology to the primer source sequence are included in the results page. This provides an option to examine other closely resembling sequences, if any, from the NRDB (non-redundant database). The non-BLAST version of ExPrimer is a one-step process that does not involve sequence analysis of primers. In all versions, the best primer pair(s) possible is/are displayed along with respective scores. All primer pairs considered for computation are listed separately with similar details.

ExPrimer could also be used to obtain primers from all the E-E-jns of the transcript instead of getting primer pairs based on one particular exon-junction and the product length.

IMPLEMENTATION

The ExPrimer program is written in PERL/CGI and runs on the Linux server located at the Institute of Bioinformatics and Applied Biotechnology (IBAB), Bangalore, India. The tool can be run on any operating system including Windows, Linux and UNIX, and can be viewed on any browser though it is best viewed on Internet Explorer 5.0.

Fig. 1

Schematic representation of the objective and function of the ExPrimer: Boxes on the mRNA indicate the source sequences for the left and right primers, respectively. As an example, the junction (E-E-jn 3) between exons 3 and 4 is shown as the source of the right primer while the left primer is on exon 2.

The authors thank Vidyendra Sadanandan for his help in writing the ExPrimer script. The work was done at IBAB. The authors received financial support from IBAB’s internal funds.