Description

The Exon PCR Primers track displays computationally designed primer pairs for PCR amplification of individual exons across all protein-coding genes. Each track item represents a complete PCR reaction designed to amplify a single exon, with primer locations highlighted as blocks within the amplicon span. This track is designed to facilitate exon-specific PCR experiments, including mutation screening, expression analysis, and targeted sequencing applications.

Display Conventions

PCR products are named using the format: {transcript_id}_exon{number}_PCR

Example: NM_001001130_exon3_PCR

Each PCR product contains comprehensive metadata accessible by clicking on track items:

Field Description Example
forwardSeq DNA sequence of the forward primer (5' → 3') ATGCGATCGTAGCATGC
forwardTm Melting temperature of forward primer (°C) 59.8
forwardGc GC content of forward primer (%) 52.4
reverseSeq DNA sequence of the reverse primer (5' → 3') GCATGCTACGATCGCAT
reverseTm Melting temperature of reverse primer (°C) 60.2
reverseGc GC content of reverse primer (%) 58.8
transcript RefSeq transcript identifier NM_001001130
geneSymbol HGNC gene symbol GAPDH
exonNum Exon number within the transcript 3
productSize Expected PCR amplicon size (bp) 185

Applications

Research Applications

Clinical Applications

Important Considerations

Data Access

Table Browser

The complete dataset can be accessed through the UCSC Table Browser:

  1. Navigate to the Table Browser
  2. Select "Exon PCR Primers" from the track dropdown
  3. Choose desired output format (BED, GTF, or custom)
  4. Apply region or gene-based filters as needed

API Access

Programmatic access is available through the UCSC REST API:

https://api.genome.ucsc.edu/getData/track?genome=hg38;track=exonPrimers;chrom=chr1;start=1000000;end=2000000

Methods

Primers are designed using Primer3 with the following default parameters:

Parameter Default Value Description
Primer Length 18-25 bp (optimal: 20 bp) Length range for primer oligonucleotides
Melting Temperature 57-63°C (optimal: 60°C) Target Tm for primer annealing
Product Size 100-300 bp Expected PCR amplicon length
Flanking Distance 500 bp Sequence context around each exon
Max Self-Complementarity 8 bp Maximum self-annealing allowed
Max 3' Self-Complementarity 3 bp Maximum 3' end self-annealing
Max Pair Complementarity 8 bp Maximum primer-dimer formation

Computational Pipeline

The primer design pipeline consists of the following steps:

1. Input Processing

2. Sequence Extraction

3. Primer Design

4. Quality Control

5. Output Generation

Limitations

Data Sources and Updates

Technical Details

File Formats

Software used

References

Primer3 Software:
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3--new capabilities and interfaces. Nucleic Acids Research 40(15):e115. doi: 10.1093/nar/gks596

ExonPrimer:
Written in the early 2000s by Tim Strom, the Exonprimer website inspired this track. The tool used to be available at http://ihg.gsf.de/ihg/ExonPrimer.html, but the server seems to be offline now.