Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2001-04-19
2004-11-09
Allen, Marianne P. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120
Reexamination Certificate
active
06816789
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of chromatography.
BACKGROUND OF THE INVENTION
Chromatography refers to a broad range of physical methods used to separate and analyze complex mixtures. The process of a chromatographic separation takes place within a chromatography column. A solvent, either a liquid or a gas depending on the type of chromatography process employed, moves through the column and carries the mixture to be separated. As the sample mixture flows through the column, its different components will adsorb to varying degrees. The differential rates of migration as the mixture moves over adsorptive materials provide separation for components in the mixture, since different components will elute from the chromatography column at different times. A detector measures the concentration/quantity of chemical or biological components that elute from the column.
A chromatogram is a chart that shows the detected quantity or concentration of various materials eluted from the column at different times. Different peaks on the chromatogram correspond to different components in the sample mixture. The size, shape, and/or position of peaks in the chromatogram can be used to help identify the various components in the mixture.
Chromatography may be used for many types of chemical/biological analysis and separation. For example, Denaturing High Pressure Liquid Chromatography (DHPLC) is routinely used to detect sequence variations in small sections of DNA. The technique is applied to samples in which a specific DNA fragment has been amplified by Polymerase Chain Reactions. The sample is analyzed by HPLC at a temperature at which the DNA fragment is close to denaturing, at which point the chromatographic behavior changes drastically depending on the thermal stability of each fragment. If the amplified DNA fragment exhibits a sequence variation, it will denature more readily than a non-variant fragment would, and the resulting chromatogram may be noticeably different. Samples without a sequence variation, or homozygous samples with a sequence variation will have complementary DNA strands and are referred to as “homoduplex”. Samples which are heterozygous for a sequence variation will form thermally less stable “heteroduplexes”, in which the DNA strands are slightly mismatched.
FIG. 1
shows a portion of an example chromatographic trace
102
resulting from DNA analysis. A typical DHPLC chromatogram
102
contains a main peak
104
corresponding to leftover PCR reagents and PCR byproducts, followed by a region with one or more peaks corresponding to the DNA fragment analyzed. This region may be followed by one or more peaks resulting from the cleaning phase, in which the HPLC system gets ready for the next analysis. To detect sequence variations in the DNA mixture, analysis is performed upon the region
106
of the chromatographic trace
102
that contains the peak corresponding to the variant gene sequence. Chromatographic trace
108
is a magnified view of chromatogram
102
, showing the region of interest
106
having a peak
110
. Normal DNA typically corresponds to a recognizable trace pattern in region of interest
106
, while variant DNA would contain a trace pattern different from the “normal” trace pattern. Thus, the particular pattern or size of the chromatographic trace in a specified region of interest can be used to separate variant DNA mixtures from normal DNA mixtures.
One approach to chromatogram classification, such as classification of chromatograms to identify the presence of sequence variations in DNA, is the “qualitative analysis” of chromatographic traces. Qualitative analysis generally refers to the analysis of chromatograms based upon the type or shape of features in the chromatographic trace. A common approach to using qualitative analysis is to perform visual examination and comparison of traces to one or more reference traces. In the example of
FIG. 1
, the shape of peak
110
in chromatogram
108
could be visually compared to that same region of a reference chromatogram to determine if chromatogram
108
corresponds to variant DNA. However, qualitative analysis involving visual examination is typically performed as a manual process that is often time-consuming and very subjective. Moreover, this type of approach is subject to a range of human errors.
An alternate approach is to employ “direct quantitative analysis” to classify chromatograms. The quantitative analysis approach may perform classification based upon, for example, the retention time, peak area, or number of peaks in a chromatogram trace. If an algorithm is used to count peaks in the chromatographic trace, then comparison can be made between the chromatogram of the DNA mixture being analyzed and the chromatogram of normal DNA based upon the number of peaks appearing in a region of interest in the chromatographic trace. However, a significant drawback with the direct quantitative analysis approach is that DNA sequence variations can result in changes to peak shape, rather than to the number of peaks, peak area, retention time, or other measures of direct quantitative analysis. Therefore, this approach may fail to adequately identify certain types of DNA sequence variations that affect the peak shape in chromatographic traces, particularly when DHPLC is used.
Thus, there is a need for an improved system and method to analyze chromatograms.
SUMMARY OF THE INVENTION
A method and system for chromatogram analysis is disclosed. An aspect of an embodiment of the invention provides a method for reducing each chromatogram to a data set that can be compared to another such data set, producing a comparison result that indicates the similarity or dissimilarity of the two chromatograms. In an embodiment, the present invention provides an automated system and method for identifying DNA sequence variations through chromatogram analysis. An embodiment provides a method and system for automated qualitative analysis and classification of chromatograms. One embodiment of the present invention also provides a user interface to display results of chromatogram analysis, which quickly and efficiently illustrates which samples are dissimilar or similar to reference chromatograms.
An object of the present invention is to provide a novel system and method to effectively and efficiently analyze and compare chromatograms. Another object of the invention is to provide a method and system for comparing and identifying variant DNA. Yet another object of the invention is to provide an interface for presenting chromatogram analysis results. These and other objects, advantages, and features of the invention will be apparent to those skilled in the art upon inspection of the specification, drawings, and claims.
REFERENCES:
patent: 5121443 (1992-06-01), Tomlinson
patent: 5905192 (1999-05-01), Wikfors et al.
patent: 6195449 (2001-02-01), Bogden et al.
patent: 0 296 781 (1988-12-01), None
patent: 0 969 283 (2000-01-01), None
Excoffier, et al., entitled “Faster Quantitative Evaluation of High-Performance Liquid Chromatography—Ultraviolet Diode-Array Data by Multicomponent Analysis”, published in Journal of Chromatography, 631, (1993), pp. 15-21.
Oefner, et al., entitled “Comparative DNA Sequence by Denturing High Performance Liquid Chromatography (DHPLC)”, published in American Journal of Human Genetics 57 (1995) A266.
Allen Marianne P.
Bingham & McCutchen LLP
Mahatan Channing S.
Varian Inc.
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