Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-13
2002-03-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S287200, C210S198200, C210S635000, C210S656000, C702S019000, C702S020000, C702S022000
Reexamination Certificate
active
06355417
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method and system for improved band array displays of double stranded nucleic acid separations. More specifically, the invention is directed toward producing linear array displays of bands representing separated double stranded nucleic acid fragments.
BACKGROUND OF THE INVENTION
Mixtures of double stranded nucleic acid fragments are separated for numerous and diverse reasons ranging from forensic investigations to gene mapping. The traditional and most widely used method for separating mixtures of DNA and other double stranded nucleic acid fragments is slab gel electrophoresis (GEP). Separation of double stranded nucleic acid fragment mixtures by this classical method produces a linear array of bands, wherein each band in the array represents a separated double stranded nucleic acid component of that mixture. Since many mixtures are typically separated and analyzed simultaneously in separate lanes on the same gel slab, a parallel series of such linear arrays of bands is produced. In principle, this is a highly desirable display format because it permits the observer to readily compare many separated mixtures simultaneously. The presence or absence of any band in one linear array compared to another is easily discernible if the separations are visible on the display.
However, in practice, GEP display methodology suffers from serious deficiencies which are inherent in the method.
Bands are often curved rather than straight, their mobility and shape can change across the width of the gel and lanes and bands can mix with each other. The sources of such inaccuracies stem from the lack of uniformity and homogeneity of the gel bed, electroendosmosis, thermal gradient and diffusion effects, as well as host of other factors. Inaccuracies of this sort are well known in the GEP art and can lead to serious distortions and inaccuracies in the display of the separation results. In addition, the band display data obtained from GEP separations is not quantitative or accurate because of the uncertainties related to the shape and integrity of the bands. True quantitation of linear band array displays produced by GEP separations cannot be achieved, even when the linear band arrays are scanned with a detector and the resulting data is integrated, because the linear band arrays are scanned only across the center of the bands. Since the detector only sees a small portion of any given band and the bands are not uniform, the results produced by the scanning method are not accurate and can even be misleading.
Linear band arrays representing components of double stranded nucleic acid mixtures which have been separated by GEP have been visualized by a variety of methods, including fluorescence, direct visualization by use of a chemical stain, by adding a dye to the gel which makes the bands visible, or by tagging the DNA with radioactive P-32 before GEP separation, followed by autoradiography. These visualization methods produce a display consisting of parallel linear arrays of bands, which is a direct hard copy representation of the gel slab itself. Separation displays produced in this manner can be distorted and inaccurate because the margins of bands so displayed often are fuzzy and diffuse, rather than being sharply defined. In the autoradiography technique, for example, the radiation emitted from the separated nucleotide components in each band is omnidirectional. This causes the exposure area of the film in contact with the gel slab to be greater than that represented by the actual band dimensions, resulting in a broadened and fuzzy display.
Analyzing adjacent bands in such displays can be a serious problem, especially when there is a large difference in the relative concentration of double stranded nucleic acid present in each band. In such a case the stronger band can obscure the weaker band and the latter may not be visible. Since the displayed data is fixed, it cannot be enhanced, optimized or manipulated, and important information can often go unnoticed. The only way to improve a defective or inadequate separation display is to re-run the separation using a more dilute sample or weaker stain. This is extremely time consuming since gel electrophoresis separations can take up to five hours or more.
A clear need, therefore, exists for an improved and flexible band array display format for double stranded nucleic acid separations in general, and DNA and RNA separations in particular, which can be electronically optimized, quantitated, and stored.
BRIEF SUMMARY OF THE INVENTION
In a first aspect the invention provides a method of representing double stranded nucleic acid fragments which have been separated by Matched Ion Polynucleotide Chromatography as an array of bands, the method comprising providing a digitized signal corresponding to the double stranded nucleic acid fragments in the fractions; and converting the digitized signal into an of array of bands. In a second aspect the invention provides an apparatus for representing double stranded nucleic acid fragments which have been separated by Matched Ion Polynucleotide Chromatography as an array of bands, the apparatus comprising acquisition means for acquiring a digitized signal, the digitized signal corresponding to the double stranded nucleic acid fragments in the fractions, conversion means, for converting the digitized signal to an array of bands corresponding to the double stranded nucleic acid fragments in the fractions, and display means for displaying the array of bands.
In a preferred embodiment of the invention, an analog signal output from the Matched Ion Polynucleotide Chromatography separation process is analog-to-digital (A/D) converted and the digitized signal is input to a computer. In the computer, the digitized signal is converted to a linear array of bands which may be displayed on a video display terminal (VDT), printer or other output device.
The bands may be displayed as lines or rectangles of fixed width. The intensity and/or color of a band may correlate to the amount of double stranded nucleic acid in the respective fraction or the respective double stranded nucleic acid fragment above a user selected threshold level at a corresponding point in the digitized signal.
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Maniatis et al Molecular Cloning A Laboratory Manual see p. 6.2-6.19, 1989.*
P. R. Haddad and P. E. Jackson “Ion Chromatography”; Elsevier NY: 1990 p. 100.
Gjerde Douglas T.
Taylor Paul D.
Horlick Kenneth R.
Siew Jeffrey
Transgenomic Inc.
Walker William B.
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