Part 1: Sequencing by CE
- CE has replaced conventional electrophoresis, significantly increasing the speed of DNA sequencing.
- Capillaries are typically 50 µm in inner diameter, about 30 cm in length, and made from high-purity fused silica.
- The small inner diameter results in excellent thermal properties, reducing Joule heating to negligible levels.
- Allows the use of extremely high electric fields for rapid separations of DNA sequencing fragments.
- Fused silica capillaries are highly flexible and easily incorporated into automated instruments, facilitating advanced sequencing.
Part 2: Capillary Array Electrophoresis
- CAE offers superior performance to conventional slab-gel electrophoresis.
- A single-capillary instrument does not offer significant advantages compared to a multilane slab-gel system.
- An array of capillaries is necessary to obtain throughput comparable to conventional electrophoresis systems.
- The first report of CAE for DNA sequencing appeared in 1990.
- Scanning a detector across the capillary array for DNA detection suffers from a limited duty cycle period.
Part 3: Continuous Capillary Monitoring
- Capillary array sequencers rely on continuous monitoring of each capillary.
- Instruments from H. Kambara at Hitachi and another group use a sheath-flow cuvet to simultaneously monitor fluorescence from a linear array of capillaries.
- One laser beam simultaneously illuminates samples migrating from all capillaries.
- An optical system images the fluorescence onto a CCD camera or an array of photodiodes.
- The instrument’s duty-cycle approaches 100%.
Part 3.1 Single Capillary Sheath-Flow Detection
- Originally developed for flow cytometry.
- A dilute suspension of fluorescently labeled cells is pumped into the center of a flowing sheath stream under laminar flow conditions.
- The fluorescence signal from each cell is measured with very low background signal from scattered laser light.
- Flow cytometry operates at high flow rates to process large numbers of cells with high precision.
- The sheath-flow cuvet was used as a high-sensitivity detector for fluorescent dyes.
- Single fluorescent molecules can be detected as they pass through the focused laser beam.
- The cuvet is used as a detector in CE due to its outstanding detection performance and small probe volume.
- The separation capillary is inserted into a square flow chamber.
- Sheath buffer surrounds the sample stream as it migrates from the capillary.
- A low-power laser beam forms a spot in the cuvet where fluorescence is detected.
- Fluorescence is collected, filtered to reduce scattered laser light, and detected with a photomultiplier tube.
- The flow chamber is held in a stainless-steel fixture completing the electrophoresis circuit.
Part 3.1.1 Sensitivity of Sheath-Flow Cuvet Detection
- Used as a detector for CE analysis of zeptomole amounts of fluorescently labeled amino acids, peptides, proteins, monosaccharides, oligosaccharides, and oligonucleotides.
- Detection limits are routinely in the y octomole range.
- Single molecules of β-phycoerythrin have been detected after CE separation.
- The first application of CE with sheath-flow detection for DNA sequencing fragments was reported in 1990.
- The first four-color sequencing application was reported in 1991.
- The sheath-flow cuvet has proven reliable for generating long sequencing read-lengths with high-temperature electrophoresis separation.
Part 3.2 Capillary Array Sheath-Flow Detection
Development:
- JianZhong Zhang developed a capillary array DNA sequencer based on a sheath-flow cuvet as part of his PhD thesis.
- Simultaneously, H. Kambara’s group developed a similar instrument.
Instrument Design:
- A linear array of capillaries is inserted into a rectangular sheath-flow cuvet.
- Sheath fluid is pumped through the interstitial space between the capillaries, entraining the DNA sequencing fragments as discrete streams, with one stream per capillary.
- A laser beam is focused into the cuvet, skimming beneath the capillary tips, exciting fluorescence from all capillaries simultaneously.
- The detector generates fluorescent spots, each spot beneath a capillary, separated by the outer diameter of the capillary.
Fluorescence Detection:
- A high numerical aperture lens collects the fluorescence and images each fluorescent spot onto a discrete photo-detector.
- Initially, fiber-optic coupled avalanche photodiodes in photon counting mode were used.
- More recently, a CCD camera is used to image the fluorescence.
- The laser beam traverses the sample streams from each capillary with negligible loss of power.
2.4. Capillary Spacing
Importance of Even Spacing:
- Even spacing of capillaries ensures uniform hydrodynamic flow of the sheath fluid.
- Uneven spacing causes variable sheath-flow rates, leading to non-uniformly spaced fluorescent spots and difficult alignment of detectors.
- Capillary spacing needs to be within ~5 µm to ensure even spacing of fluorescence spots.
Methods to Ensure Even Spacing:
Intimate Contact Method:
- Capillaries are in intimate contact within the cuvet, with center-to-center spacing determined by the outer diameter of the capillary.
- Typically held to within 1 µm across a 100-m long reel of capillary.
- Variations in reel-to-reel dimensions can exceed 10 µm.
- A wedge-shaped cuvet with tapered walls forces the capillaries into contact as they are inserted.
Finger Micromachining Method:
- Fingers are micromachined in the inner wall of the cuvet.
- These raised features hold the capillaries on uniform centers.
Reference: Capillary Electrophoresis of Nucleic Acids by Keith R. Mitchelson Jing Cheng