Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1999-11-24
2001-10-30
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S172000, C422S082080, C356S072000, C356S073000
Reexamination Certificate
active
06309886
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the problem of increasing sample throughput and analysis for fluorescence based flow cytometry, and, more particularly, to DNA fragment sizing using flow cytometry as the flow-based luminescence system.
BACKGROUND OF THE INVENTION
DNA fragment sizing is one of the most widely used analytical techniques in the biological, biomedical, and forensic sciences, and is becoming increasingly important in the environmental sciences, as well. Techniques for DNA sequencing, DNA fingerprinting, restriction mapping, and genotyping all rely on the ability to accurately determine the size distribution of DNA fragments in a multicomponent solution. Gel electrophoresis is currently the standard analytical tool for sizing DNA. Conventional gel electrophoresis possesses single base pair (bp) sizing resolution for 10 to ~700 bp DNA and can measure fragments as large as a few tens of kilobase pairs (kbp) with a sizing resolution of ~10%. Fragments ranging from tens to hundreds of kbp can be sized using pulsed field gel electrophoresis (PFGE) with ~10% sizing resolution. These techniques provide accurate information that is widely accepted. However, there are increasing demands for DNA sizing methods with higher speed and better sensitivity than gel electrophoresis can provide. To meet these demands, a number of groups are developing novel approaches to DNA sizing, based on such techniques as single molecule fluorescence burst detection, capillary electrophoresis, optical mapping, mass spectrometry, and atomic force microscopy.
Fluorescence burst detection of single DNA molecules labeled with fluorescent intercalating dyes has shown significant improvements in the speed and sensitivity of DNA fragment sizing compared to gel electrophoresis, particularly for the sizing of DNA fragments in the size range of hundreds of bp to hundreds of kbp. The quantity of fluorescent dye intercalated into a DNA fragment is directly proportional to its length. Thus, assuming that each fragment experiences the same excitation intensity, the number of fluorescence photons detected per fragment (burst size) is a direct measure of the DNA fragment size. The DNA fragment sizing technique that has been developed at Los Alamos National Laboratory, referred to as single molecule flow cytometry (SMFC), has been demonstrated to size femtogram (fg) quantities of >300 bp to 425 kbp DNA in a complex mixture of DNA fragment sizes after several minutes of data acquisition and analysis. This is compared to the nanogram (ng) quantities of DNA and the analysis tirhes of several hours required for DNA fragment sizing by gel electrophoresis. For fragments >20 kbp, the sizing resolution obtained by SMFC is similar to PFGE, and the accuracy is better (2% versus 10% uncertainty). In addition, the burst sizes are linear with fragment size. DNA fragment sizing by SMFC has recently been applied to the characterization of P
1
artificial chromosome (PAC) clones, the analysis of PCR fragments, and bacteria fingerprinting.
In spite of the substantial improvements in the speed and sensitivity of DNA fragment sizing by SMFC, there are numerous applications in the biomedical and environmental sciences that would benefit greatly from even higher sample throughput for sizing trace quantities of DNA present in large sample volumes or for rapidly characterizing large numbers of samples. The sample throughput provided by conventional SMFC is limited to a maximum burst detection rate of ~100 fragments per second by the finite transit time (~2 ms) of the DNA fragments through the detection region needed to acquire a sufficient number of photoelectrons per fragment for adequate counting statistics. Increasing the sample delivery rate beyond ~100 fragments per second results in a high incidence of events corresponding to double occupancy of the detection region, while decreasing the transit time reduces the burst sizes and thus degrades the resolution. The laser irradiance cannot be increased to compensate for a shorter transit time because the fluorescence becomes optically saturated. Optical saturation is the limited fluorescence rate due to ground state depletion caused by finite excited state lifetimes.
The present invention is directed to a new approach to detecting particles in biochemistry that, in its present configuration, has demonstrated the measurement of thousands of DNA fragments per second, resulting in more than an order of magnitude increase in the sample throughput compared to conventional SMFC. The volumetric flow rate of the analyte solution was increased to 2.5 &mgr;l/min, compared to a typical value of ~0.2 &mgr;l/min for the conventional approach. This increase in sample throughput was accomplished by combining SMFC with parallel fluorescence imaging detection using a charge coupled device (CCD) camera. Whereas conventional SMFC monitors the fluorescence from a single detection volume, defined by the interaction of a tightly focused laser beam and the image of an aperture at the laser focus, this new approach simultaneously monitors thousands of detection volumes, defined by the number of pixels in the CCD array.
This technique has been applied to the detection and sizing of individual DNA-dye complexes in the 7 to 154 kbp size range, although much larger or smaller fragments can be analyzed in this way. As with fragment sizing by conventional SMFC, the burst sizes were linear with DNA fragment size. Unlike conventional SMFC, data sufficient to determine the DNA fragment size distribution were acquired in less than 10 seconds. At present, the smallest DNA-dye complex that can be detected with a signal-to-noise ratio of ~2 is 1.86 kbp. A measurement rate as high as 10
5
DNA fragments per second can be achieved by using a faster detector than the one used in the initial experiments, while still maintaining this same level of sensitivity.
Various objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
As embodied and broadly described herein, the present invention includes apparatus and method for imaging multiple fluorescent sample particles in a flow channel. A flow channel defines a flow direction for samples in a flow stream and has a viewing plane perpendicular to the flow direction. A laser beam is formed as a ribbon having a width effective to cover the viewing plane. Imaging optics are arranged to view the viewing plane to form an image of the multiple fluorescent sample particles in the flow stream and a camera records the image formed by the imaging optics.
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Ambrose W. Patrick
Goodwin Peter M.
Grace W. Kevin
Jett James H.
Keller Richard A.
Snay Jeffrey
The Regents of the University of California
Wilson Ray G.
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