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
- Track Items: Each item represents one PCR amplicon spanning from the forward primer to the reverse primer
- Color: All PCR products are displayed in green (RGB: 0,128,0)
- Blocks: Two thick blocks within each item indicate the exact locations of the forward and reverse primers
- Strand: No strand indicators are shown (strand = ".")
- Score: Represents the average melting temperature (Tm) of both primers × 10
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
- Mutation Screening: PCR amplification of exons for Sanger sequencing or variant detection
- Expression Analysis: RT-PCR analysis of exon inclusion/exclusion patterns
- Targeted Sequencing: Primer design for custom amplicon sequencing panels
- Cloning: Exon-specific amplification for molecular cloning applications
- Functional Studies: Generation of exon-specific constructs for functional analysis
Clinical Applications
- Diagnostic PCR: Disease gene mutation screening in clinical samples
- Pharmacogenomics: Analysis of drug metabolism gene variants
- Genetic Testing: Targeted analysis of known pathogenic variants
Important Considerations
- Validation Required: All primers should be experimentally validated before use
- Specificity: Check primer specificity using BLAST or similar tools
- Polymorphisms: Consider known SNPs/variants that may affect primer binding
- Splice Variants: Primers may not amplify all transcript isoforms
- Pseudogenes: Be aware of potential cross-amplification with pseudogenes
Data Access
Table Browser
The complete dataset can be accessed through the UCSC Table Browser:
- Navigate to the Table Browser
- Select "Exon PCR Primers" from the track dropdown
- Choose desired output format (BED, GTF, or custom)
- 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
- UCSC genePred format gene annotations are parsed to extract exon coordinates
- Transcript information including gene symbols are retained
- Only protein-coding transcripts are processed
2. Sequence Extraction
- Genomic sequences are retrieved from 2bit genome files
- Flanking sequences (default 500 bp) are added to each exon
- Repetitive sequences are masked using standard genome masks
3. Primer Design
- Primer3 is executed in batch mode for computational efficiency
- Target regions are set to exon boundaries within the flanking sequence
- Primer pairs are optimized for uniform melting temperatures and minimal secondary structure
4. Quality Control
- Primers failing quality criteria are excluded
- Genomic coordinates are calculated and validated
- Primer specificity is assessed computationally
5. Output Generation
- Results are formatted as BED12 files with metadata
- BigBed files are generated for genome browser display
- Comprehensive metadata is embedded in extra fields
Limitations
- Computational Design: Primers are designed computationally and may require experimental optimization
- Single Isoform: Primers target the canonical transcript and may not amplify all splice variants
- Repetitive Regions: Exons in highly repetitive regions may lack suitable primers
- Polymorphisms: Common genetic variants may affect primer efficiency
- Species Specificity: Primers are designed for human sequences only
Data Sources and Updates
- Gene Annotations: UCSC Gencode V48
- Genome Sequence: Reference genome assemblies (hg19, hg38)
- Update Frequency: Updated with each major Gencode annotation release
- Quality Assurance: Automated validation against current genome builds
Technical Details
File Formats
- Track Format: BigBed 12+10
- Coordinate System: 0-based, half-open intervals
- Strand Convention: No strand displayed (strand = ".")
- Color Encoding: RGB values embedded in itemRgb field
Software used
- Primer3: Primer design and optimization
- UCSC Tools: Genome sequence access and file format conversion
- Python Libraries: twobitreader for genome access
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.