Genome-scale design of PCR primers and long oligomers for DNA microarrays - PubMed (original) (raw)

Genome-scale design of PCR primers and long oligomers for DNA microarrays

Stefan A Haas et al. Nucleic Acids Res. 2003.

Abstract

During the last years, the demand for custom-made cDNA chips/arrays as well as whole genome chips is increasing rapidly. The efficient selection of gene-specific primers/oligomers is of the utmost importance for the successful production of such chips. We developed GenomePRIDE, a highly flexible and scalable software for designing primers/oligomers for large-scale projects. The program is able to generate either long oligomers (40-70 bases), or PCR primers for the amplification of gene-specific DNA fragments of user-defined length. Additionally, primers can be designed in-frame in order to facilitate large-scale cloning into expression vectors. Furthermore, GenomePRIDE can be adapted to specific applications such as the generation of genomic amplicon arrays or the design of fragments specific for alternative splice isoforms. We tested the performance of GenomePRIDE on the entire genomes of Listeria monocytogenes (1584 gene-specific PCRs, 48 long oligomers) as well as of eukaryotes such as Schizosaccharomyces pombe (5006 gene-specific PCRs), and Drosophila melanogaster (21 306 gene-specific PCRs). With its computing speed of 1000 primer pairs per hour and a PCR amplification success of 99%, GenomePRIDE represents an extremely cost- and time-effective program.

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Figures

Figure 1

Example of a text file describing two overlapping sequence tags. The line starting with ‘ID’ defines the contig on which the tag has to be placed. The following line describes the type of the tag, its orientation (+/–) and the start/end positions. Further ‘TC’ lines contain comments like the name of the corresponding gene or some description.

Figure 2

Annotations of sequences interpreted by GenomePRIDE. The Staden package allows the marking of sequence features [exons (orange boxes), repeats (green boxes), homology regions (gray boxes)] by specific tags (Table 1) sketched in (A). Based on these tags, GenomePRIDE generates an artificial subsequence of the genome that includes all sequence information relevant for the primer/oligo design for a single gene (B). This sequence consists of a concatenation of all exons flanked by upstream/downstream sequences (dashed lines) of a user-defined length (default 10 kb). In order to enable GenomePRIDE to check for secondary binding sites within the introns of the gene of interest, these intronic sequences are appended. Similarities to more distant genes are still represented by the appropriate tagging.

Figure 3

Length distribution of PCR fragments (D.melanogaster). A large fraction of the PCR fragments matches the user-defined fragment length of 500 bp. GenomePRIDE designed some fragments including intronic sequence up to a total length of ∼600 bp since a small amount of intron sequence (<15%) is tolerated. Only a few fragments are longer than 600 bp, this being caused by genes with exons shorter than the minimal fragment length of 100 bp.

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Genome-scale design of PCR primers and long oligomers for DNA microarrays - PubMed (original) (raw)