Time-Resolved Fluorescence Imaging of Slab Gels for Lifetime Base-Calling in DNA Sequencing Applications
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- 作者：Suzanne J. Lassiter ; Wieslaw Stryjewski ; Benjamin L. Legendre ; Jr. ; Rainer Erdmann ; Michael Wahl ; John Wurm ; Rex Peterson ; Lyle Middendorf ; and Steven A. Soper
- 刊名：Analytical Chemistry
- 出版年：2000
- 出版时间：November 1, 2000
- 年：2000
- 卷：72
- 期：21
- 页码：5373 - 5382
- 全文大小：308K
- 年卷期：v.72,no.21(November 1, 2000)
- ISSN：1520-6882
文摘
A compact time-resolved near-IR fluorescence imager wasconstructed to obtain lifetime and intensity images of DNAsequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which wasmounted a pulsed diode laser (repetition rate 80 MHz,lasing wavelength 680 nm, average power 5 mW), filteringoptics, and a large photoactive area (diameter 500 
m)single-photon avalanche diode that was actively quenchedto provide a large dynamic operating range. The time-resolved data were processed using electronics configuredin a conventional time-correlated single-photon-countingformat with all of the counting hardware situated on a PCcard resident on the computer bus. The microscope headproduced a timing response of 450 ps (fwhm) in ascanning mode, allowing the measurement of subnanosecond lifetimes. The time-resolved microscope head wasplaced in an automated DNA sequencer and translatedacross a 21-cm-wide gel plate in ~6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. Thesampling frequency was 0.17 Hz (duty cycle 0.0017),sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic)allowed acquisition of both the intensity image and lifetimeanalysis of DNA bands migrating through the gel in realtime. Using a dual-labeling (IRD700 and Cy5.5 labelingdyes)/two-lane sequencing strategy, we successfully read670 bases of a control M13mp18 ssDNA template usinglifetime identification. Comparison of the reconstructedsequence with the known sequence of the phage indicatedthe number of miscalls was only 2, producing an errorrate of ~0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used toconstruct the decay profiles. Comparison of the lifetimebase calling to a single-dye/four-lane sequencing strategyindicated similar results in terms of miscalls, but reducedinsertion and deletion errors using lifetime identificationmethods, improving the overall read accuracy.
m)single-photon avalanche diode that was actively quenchedto provide a large dynamic operating range. The time-resolved data were processed using electronics configuredin a conventional time-correlated single-photon-countingformat with all of the counting hardware situated on a PCcard resident on the computer bus. The microscope headproduced a timing response of 450 ps (fwhm) in ascanning mode, allowing the measurement of subnanosecond lifetimes. The time-resolved microscope head wasplaced in an automated DNA sequencer and translatedacross a 21-cm-wide gel plate in ~6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. Thesampling frequency was 0.17 Hz (duty cycle 0.0017),sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic)allowed acquisition of both the intensity image and lifetimeanalysis of DNA bands migrating through the gel in realtime. Using a dual-labeling (IRD700 and Cy5.5 labelingdyes)/two-lane sequencing strategy, we successfully read670 bases of a control M13mp18 ssDNA template usinglifetime identification. Comparison of the reconstructedsequence with the known sequence of the phage indicatedthe number of miscalls was only 2, producing an errorrate of ~0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used toconstruct the decay profiles. Comparison of the lifetimebase calling to a single-dye/four-lane sequencing strategyindicated similar results in terms of miscalls, but reducedinsertion and deletion errors using lifetime identificationmethods, improving the overall read accuracy.