Capillary Electrophoresis¶
Overview¶
Capillary electrophoresis (CE) separates DNA fragments by size using an electric field within a narrow, liquid-filled capillary. Unlike traditional agarose gel electrophoresis — which separates fragments by migration through a porous gel matrix — CE passes fragments through a polymer-filled capillary while detecting fluorescently labeled molecules by laser-induced fluorescence as they pass a detection window.
CE provides base-pair resolution of DNA fragments, making it the method of choice for applications that require precise sizing: distinguishing CHD-Z and CHD-W amplicons that may differ by only 10–30 base pairs, and scoring microsatellite alleles for individual DNA fingerprinting and parentage verification.
SENO operates two CE instruments supporting high-throughput processing for sexing, fingerprinting, and research applications. CE analysis is performed on PCR products amplified using fluorescently labeled primers as described in our PCR Technology section.
Principle of Operation¶
CE separates DNA fragments based on their electrophoretic mobility through a sieving polymer. Smaller fragments migrate faster and reach the detector earlier. A laser excites fluorescent dyes attached to the labeled primers, and the emitted fluorescence is recorded by a CCD camera. An internal size standard (labeled with a spectrally distinct dye) is co-injected with each sample, enabling fragment sizing by interpolation.
| Component | Function |
|---|---|
| Capillary (36–50 cm length, 50 µm ID) | Separation channel filled with linear polymer matrix |
| Polymer matrix | Sieving medium — separates fragments by size |
| Running buffer | Provides electrical continuity (1× or 10× Genetic Analyzer Buffer) |
| Laser (488 nm / 514 nm) | Excites fluorescent dyes on labeled primers |
| CCD camera | Records fluorescence emission across multiple spectral channels |
| POP-4™ or POP-7™ polymer | Commercial sieving polymers optimized for different size ranges |
Advantages Over Gel Electrophoresis¶
| Feature | Agarose Gel Electrophoresis | Capillary Electrophoresis |
|---|---|---|
| Resolution | ± 10–20 bp | ± 1–2 bp (single base pair) |
| Throughput | 20–50 samples per gel | 96–384 samples per run |
| Sample volume required | 8–10 µL PCR product | ~1 µL of diluted PCR product |
| Automation | Manual (gel pouring, loading, imaging) | Fully automated (plate loading → data export) |
| Data format | Image (gel photo) | Digital (electropherogram: peaks × sizes) |
| Quantification | Visual estimation (band intensity) | Precise (peak area, peak height, RFU) |
| Multiplex detection | Separate gels or co-migrating bands | Multi-color detection (4–6 dyes per capillary) |
| Reproducibility between runs | Moderate | High (± 0.5 bp sizing precision) |
| Hands-on time per 96 samples | ~45 min (gel prep + loading + imaging) | ~15 min (plate setup + instrument start) |
| Data export to LIMS | Manual (gel image annotation) | Automated (allele tables, peak lists) |
SENO's CE Applications¶
DNA Sexing Fragment Analysis¶
CE provides precise sizing of CHD-W and CHD-Z amplicons, resolving fragments that may appear as a single band on agarose gel. This is particularly valuable for species where the CHD-Z and CHD-W intron lengths are similar.
| Parameter | CE Sexing | Gel Sexing |
|---|---|---|
| Fragment size accuracy | ± 1 bp | ± 10–20 bp |
| Detection of small CHD-W/Z differences | Down to 5 bp difference | Typically needs ≥ 20 bp |
| Automated allele calling | Yes (GeneMapper) | No (manual band scoring) |
| Sample volume requirement | 1 µL of 1:20 dilution | 8 µL undiluted |
| Turnaround for 96 samples | ~3 hours (instrument) + 30 min (analysis) | ~2 hours (gel) + 30 min (imaging/analysis) |
In practice, CE is used as a confirmatory method for difficult sexing cases (where gel results are ambiguous) and as the primary method for species known to have small CHD intron length differences.
DNA Fingerprinting (Microsatellite Genotyping)¶
DNA fingerprinting at SENO uses a panel of 12 microsatellite loci for individual identification and parentage verification. The discriminatory power of this panel is exceptionally high (DP > 0.9999).
| Locus | Repeat Motif | Size Range (bp) | Fluorescent Label | Alleles Observed | He (Expected) |
|---|---|---|---|---|---|
| Pgi01 | (CA)₁₉ | 180–220 | FAM (blue) | 12 | 0.81 |
| Pgi02 | (GT)₂₃ | 140–180 | HEX (green) | 15 | 0.85 |
| Pgi03 | (CA)₁₅ | 200–240 | FAM (blue) | 10 | 0.78 |
| Pgi04 | (ATCT)₁₂ | 160–200 | TAMRA (yellow) | 8 | 0.72 |
| Pgi05 | (CA)₁₇ | 120–160 | FAM (blue) | 14 | 0.83 |
| Pgi06 | (GT)₁₈ | 220–260 | HEX (green) | 11 | 0.80 |
| Pgi07 | (CA)₂₁ | 170–210 | FAM (blue) | 13 | 0.82 |
| Pgi08 | (CA)₁₆ | 130–170 | TAMRA (yellow) | 10 | 0.77 |
| Pgi09 | (GT)₁₅ | 240–280 | HEX (green) | 9 | 0.75 |
| Pgi10 | (ATCT)₁₀ | 150–190 | FAM (blue) | 7 | 0.69 |
| Pgi11 | (CA)₁₄ | 280–320 | HEX (green) | 12 | 0.81 |
| Pgi12 | (GT)₂₀ | 190–230 | TAMRA (yellow) | 11 | 0.79 |
- Combined Power of Discrimination (PD): > 0.9999
- Combined Exclusion Probability (PE, single parent): > 0.999
- Combined Exclusion Probability (PE, both parents): > 0.99999
Product Quality Verification¶
CE fragment analysis is used to confirm PCR product specificity:
| Purpose | PCR Product | CE Check | Outcome |
|---|---|---|---|
| Verify CHD amplicon sizes | Sexing PCR | Fragment sizing ± 1 bp | Confirms expected Z/W pattern |
| Check for primer-dimer | Any PCR | Peak < 80 bp confirmed as primer-dimer | Excluded from analysis |
| Confirm multiplex fidelity | Multiplex PCR | Expected peak pattern in each dye channel | All loci amplified |
CE Workflow¶
| Step | Action | Responsible | Duration |
|---|---|---|---|
| 1 — PCR with labeled primers | Amplify targets using fluorescently labeled forward primers | Technician | 1.5–2 h |
| 2 — Post-PCR cleanup (optional) | Remove unincorporated primers and dNTPs | Technician | 15 min |
| 3 — Sample dilution | Dilute PCR product (1:10–1:40 in Hi-Di formamide) depending on yield | Technician | 5 min |
| 4 — Add internal size standard | Add GeneScan™ 500 LIZ™ or equivalent to each sample | Technician | 5 min |
| 5 — Denaturation | 95°C for 3 min, then snap-cool on ice | Technician | 5 min |
| 6 — Plate setup | Transfer samples to 96- or 384-well plate, seal with septa | Technician | 10 min |
| 7 — Instrument run | Load plate, select assay, start CE run | Technician | 25–60 min (per injection) |
| 8 — Data export | Automated size calling and allele detection software processes raw data | Software | 10–15 min |
| 9 — Manual review | Technician reviews electropherograms, adjusts bin assignments | Senior Tech | 15–30 min |
| 10 — LIMS upload | Final allele calls exported to LIMS for report generation | Software | 5 min |
CE Instrumentation¶
SENO operates two capillary electrophoresis instruments for routine testing and R&D.
| Instrument | Capillary Count | Sample Capacity | Run Time (96 samples) | Applications |
|---|---|---|---|---|
| Applied Biosystems 3500 Genetic Analyzer | 8 capillaries | 96-well plates | ~45 min | Sexing fragment analysis, fingerprinting |
| Applied Biosystems SeqStudio Flex | 4 capillaries | 96-well plates | ~60 min | Fingerprinting, research, validation |
Both instruments support multi-color detection (6-dye capability), automated polymer filling, and walk-away operation. Regular maintenance includes:
| Maintenance | Frequency | Action |
|---|---|---|
| Polymer replacement | Per run | Replace with fresh POP-4 or POP-7 |
| Buffer replacement | Per run | Fresh 1× running buffer |
| Capillary conditioning | Weekly | Conditioning run (polymer wash + electro-kinetic conditioning) |
| Capillary replacement | Every 1,500 injections or annually | When resolution degrades or signal intensity drops |
| Instrument calibration | Quarterly | Dye spectral calibration, size standard verification |
| Preventive maintenance | Bi-annual | Pump seal replacement, optical alignment check, fan filter cleaning |
Quality Controls in CE¶
| Control | Content | Expected Result | Purpose |
|---|---|---|---|
| Size standard | GS500 LIZ (35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, 500 bp) | All peaks present, within ± 0.5 bp | Sizing accuracy |
| Positive control (known genotype) | Amplified DNA from reference individual | Expected allele sizes ± 1 bp | Assay setup verification |
| Negative control | Nuclease-free water through PCR | No peaks above threshold | Contamination detection |
| Allelic ladder | Pool of known alleles for each locus | All expected allele bins called | Sizing consistency across runs |
| Blank injection | Hi-Di formamide only | No peaks | Capillary cleanliness |
Troubleshooting CE Issues¶
| Problem | Possible Cause | Solution |
|---|---|---|
| No signal in any sample | Laser off, polymer not filled, injection failure | Check instrument status; re-run with fresh polymer |
| Weak signal (low RFU) | Insufficient PCR product, over-dilution | Reduce dilution (1:10 instead of 1:40); increase injection time |
| Split peaks / doublets | Salt in sample, incomplete denaturation | Add cleanup step; re-denature at 95°C for 3 min |
| Sizing off by > 1 bp | Degraded size standard, expired polymer | Replace size standard; use fresh polymer |
| Broad peaks | Old polymer, degraded capillary | Replace polymer; check capillary life count |
| Spikes in electropherogram | Air bubbles, impurities in polymer | Degas polymer; purge capillaries |
| Pull-up between dye channels | Saturated signal in one dye channel | Reduce injection time or dilute sample further |
| Late fragment pull (differential migration) | Overloaded sample | Dilute and re-inject at lower concentration |
Cross-References¶
- PCR Technology → — PCR amplification step before CE analysis
- qPCR / Fluorescent PCR → — Alternative fluorescent detection method
- Reagent Development → — Fluorescent primer design and production
- DNA Fingerprinting — Microsatellite genotyping applications
- Parentage Verification — CE-based parentage analysis
- Laboratory Workflow — End-to-end sample processing pipeline
- Accuracy Validation — CE method performance data
Frequently Asked Questions¶
Q: Why use CE instead of agarose gel electrophoresis for sexing? A: CE provides single-base-pair resolution, enabling confident discrimination of CHD-Z and CHD-W fragments that differ by as few as 5–10 bp. Agarose gel can miss small differences, leading to incorrect sex assignment in species where CHD introns are nearly identical in length.
Q: How many microsatellite loci does SENO use for fingerprinting? A: Our standard DNA fingerprinting panel includes 12 microsatellite loci, providing a combined power of discrimination exceeding 0.9999. This means the probability of two unrelated individuals sharing the same profile is less than 1 in 10,000.
Q: What size standard does SENO use? A: We use GeneScan™ 500 LIZ™ (16 fragments from 35 to 500 bp) for most applications. For microsatellites with larger alleles (> 400 bp), we use GeneScan™ 600 LIZ™.
Q: Can CE distinguish between same-sex siblings? A: Yes. While CHD sexing identifies the genetic sex, microsatellite fingerprinting by CE distinguishes individual identity regardless of sex. Same-sex siblings will have different microsatellite profiles (unless they are identical twins, which is extremely rare in birds).
Q: How long does a CE run take? A: For the ABI 3500 (8 capillaries), a full 96-sample run takes approximately 45 minutes. For the SeqStudio Flex (4 capillaries), approximately 60 minutes. Total turnaround including plate preparation and data analysis is about 3–4 hours for 96 samples.
This document describes SENO's capillary electrophoresis methodology and applications. For CE protocol SOPs, refer to the Quality Control section. For questions about fingerprinting applications, contact [email protected].