Time resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications

被引:25
作者
Lassiter, SJ [1 ]
Stryjewski, W [1 ]
Legendre, BJ [1 ]
Erdmann, R [1 ]
Wahl, M [1 ]
Wurm, J [1 ]
Peterson, R [1 ]
Middendorf, L [1 ]
Soper, SA [1 ]
机构
[1] Louisiana State Univ, Dept Chem, Baton Rouge, LA 70803 USA
关键词
D O I
10.1021/ac000744v
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
A compact time-resolved near-IR fluorescence imager nas constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 mum) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnanosecond lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in similar to6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (witten in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of similar to0.3% (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 to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.
引用
收藏
页码:5373 / 5382
页数:10
相关论文
共 25 条
[1]   SENSITIVE FLUORESCENCE DETECTION IN CAPILLARY ELECTROPHORESIS USING LASER-DIODES AND MULTIPLEX DYES [J].
BACHTELER, G ;
DREXHAGE, KH ;
ARDENJACOB, J ;
HAN, KT ;
KOLLNER, M ;
MULLER, R ;
SAUER, M ;
SEEGER, S ;
WOLFRUM, J .
JOURNAL OF LUMINESCENCE, 1994, 62 (3-4) :101-108
[2]   High speed electronics for the detection of time-resolved fluorescence in a continuous flow system [J].
Erdmann, R ;
Ortmann, U ;
Enderlein, J ;
Becker, W ;
Wahl, M ;
Klose, E .
ULTRASENSITIVE BIOCHEMICAL DIAGNOSTICS, PROCEEDINGS OF, 1996, 2680 :176-181
[3]   Near-infrared heavy-atom-modified fluorescent dyes for base-calling in DNA-sequencing applications using temporal discrimination [J].
Flanagan, JH ;
Owens, CV ;
Romero, SE ;
Waddell, E ;
Kahn, SH ;
Hammer, RP ;
Soper, SA .
ANALYTICAL CHEMISTRY, 1998, 70 (13) :2676-2684
[4]   BETTER ESTIMATES OF EXPONENTIAL DECAY PARAMETERS [J].
HALL, P ;
SELINGER, B .
JOURNAL OF PHYSICAL CHEMISTRY, 1981, 85 (20) :2941-2946
[5]   On-the-fly fluorescence lifetime detection of dye-labeled DNA primers for multiplex analysis [J].
He, H ;
Nunnally, BK ;
Li, LC ;
McGown, LB .
ANALYTICAL CHEMISTRY, 1998, 70 (16) :3413-3418
[6]  
HUANG XC, 1992, ANAL CHEM, V64, P167
[7]   DNA SEQUENCING USING CAPILLARY ARRAY ELECTROPHORESIS [J].
HUANG, XHC ;
QUESADA, MA ;
MATHIES, RA .
ANALYTICAL CHEMISTRY, 1992, 64 (18) :2149-2154
[8]   MULTIPLE-SHEATHFLOW CAPILLARY ARRAY DNA ANALYZER [J].
KAMBARA, H ;
TAKAHASHI, S .
NATURE, 1993, 361 (6412) :565-566
[9]   An all solid-state near-infrared time-correlated single photon counting instrument for dynamic lifetime measurements in DNA sequencing applications [J].
Legendre, BL ;
Williams, DC ;
Soper, SA ;
Erdmann, R ;
Ortmann, U ;
Enderlein, J .
REVIEW OF SCIENTIFIC INSTRUMENTS, 1996, 67 (11) :3984-3989
[10]   On-the-fly fluorescence lifetime detection of labeled DNA primers [J].
Li, LC ;
He, H ;
Nunnally, BK ;
McGown, LB .
JOURNAL OF CHROMATOGRAPHY B, 1997, 695 (01) :85-92