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An analytical system based on a compact flow cytometer for DNA fragment sizing and single‐molecule detection
Author(s) -
Habbersett Robert C.,
Jett James H.
Publication year - 2004
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20042
Subject(s) - physics , laser , photomultiplier , dna , photon counting , sensitivity (control systems) , photon , optics , chemistry , materials science , analytical chemistry (journal) , detector , chromatography , biochemistry , electronic engineering , engineering
Background Previous reports have demonstrated accurate DNA fragment sizing of linear DNA fragments, from 564 to ≈4 × 10 5 bp, in a flow system. B‐phycoerythrin (B‐PE), commonly used in conventional cytometric applications that require high‐sensitivity, was the first fluorophore detected in flow at the single‐molecule level. Methods Dilute solutions of stained DNA fragments or B‐PE were analyzed in a simplified, compact flow system, with enhanced performance and lower cost, utilizing a solid‐state laser and a single‐photon sensing avalanche photodiode detector (SSAPD). Extensive data processing and display software, developed specifically for the photon‐counting data stream, extracts correlated height, width, and area features from bursts of photons due to discrete molecules passing through the sensing region in the flow channel. Results DNA fragment sizing in flow has now been demonstrated for SYTOX‐orange–stained fragments ranging in size over 3.4 orders of magnitude, from 125 to 5 × 10 5 bp. For Lambda bacteriophage DNA (λ DNA; 48.5 kbp) a CV of 1.2 % has been achieved. Analysis of a femtomolar B‐PE solution demonstrates that the bursts of photons from individual molecules can be baseline‐resolved with 0.5 mW of laser power at a signal to noise ratio (SNR) of ≈30, with ≈100 photons detected from each molecule. Conclusions A compact, low‐power, high‐sensitivity system detects DNA fragments as small as 125 bp or individual B‐PE molecules in a flowing liquid stream. Demonstrated linearity, sensitivity, and resolution indicate that <1.0 mW of laser power is optimal, permitting further miniaturization of the system and additional cost reduction. Comprehensive analytical software exploits the standard cytometric paradigm of multiple 2D graphs and gating to extract features from classes of individually analyzed biomolecules. This complete system is thus poised to engage high‐sensitivity applications not amenable to conventional flow cytometric instrumentation. © 2004 Wiley‐Liss, Inc.