Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
1999-12-17
2004-03-02
Wachsman, Hal (Department: 2857)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C702S019000, C702S030000, C436S164000, C422S082050
Reexamination Certificate
active
06701254
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for analysing one or more samples in an array of samples, preferably biomolecules, and an apparatus for carrying out these methods.
BACKGROUND ART
Improvements in laboratory techniques and practices have led to the discovery of an ever increasing number of new biomolecules. New protein purification and detection methods, for example, have allowed the detection of many possibly new proteins. Due to the large number of known biomolecules, it is now necessary to carry out molecular comparisons of newly discovered molecules to determine to what extent they are similar to or different from known molecules. To carry out definitive analyses for proteins for example it is necessary to obtain amino acid sequence information. Unfortunately, current methods and apparatus for such analyses are slow and are only able to analyse one or a few samples at one time. In order to carry out analysis of a given protein at present it is necessary to obtain the protein in substantially pure and isolated form. There is a need for methods and apparatus that can analyse one desired sample from an array of samples or be able to analyse multiple samples.
The present inventors have now realised that it is possible to develop improved methods and apparatus suitable to carry out these types of analyses.
DISCLOSURE OF INVENTION
The present invention relates generally to methods for analysing at least one sample in an array of samples by recording an image of the position of at least one sample relative to the other samples in the array and utilizing the recorded image so as to allow the analysis of the at least one sample in situ.
In a first aspect, the present invention consists of a method for analysing at least one sample in an array of samples, the method including the steps:
(a) recording an image of the position of at least one sample relative to the other samples in the array;
(b) utilizing the recorded image so as to allow the application of a reagent or a succession of reagents to the at least one sample in situ; and
(c) analysing the at least one sample for a reaction to or with the reagent(s).
In a preferred form, the samples are biomolecules selected from the group consisting of proteins, peptides, saccharides, lipids, nucleic acid molecules, complex biomolecules including glycoproteins, and mixtures thereof. The biomolecules are preferably separated by chromatography to form an array of samples. The chromatography is preferably electrophoresis, and more preferably electrophoresis is carried out in a polyacrylamide gel.
The polyacrylamide gel electrophoresis can be carried out in one dimension including isoelectric focusing, native polyacrylamide gel electrophoresis, and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Alternatively, the polyacrylamide gel electrophoresis is carried out in two dimensions with the first dimension by isoelectric focusing and the second dimension is by SDS polyacrylamide gel electrophoresis.
Preferably, the biomolecules separated by electrophoresis are transferred to a semi-solid or solid support. The solid support can be a membrane made of polyvinylidene difluoride, nitrocellulose, nylon, Teflon™, Zitex™, polypropylene, polytetrafluoroethylene, and derivatised forms thereof having one or more functional groups.
Preferably, the biomolecules transferred to semi-solid or solid support are visualised by association with a dye, fluorescent group or metal, or by association with a second biomolecule which is coupled with a third biomolecule, dye, fluorescent group or metal. The array of samples is preferably in a plane in order to assist in the recording of the image.
In a preferred embodiment of the present invention, the image is generated from a scan of the samples stained or illuminated to allow them to be visualised and the application of the reagent or reagents is carried out using a chemical printer based on an “ink jet” or similar application system where the reagent or reagents are discharged to the desired sample by the chemical printer.
It will be appreciated that the array to be manipulated may not necessarily be the array from which the image was obtained. For example, it would be possible to make multiple identical arrays of samples and use one array to obtain the image but use one or more of the multiple identical arrays to carry out the manipulations. For example in protein separation by 2 dimension polyacrylamide gel electrophoresis (2D PAGE) one separation gel may be blotted to more than one support or multiple identical separations carried out and each transferred to a support to form the identical arrays.
The analysing may be by any means known to the art. Suitable examples include use of liquid chromatograph, photoelectrical, photochemical, laser, radiochemical, and mass spectral analyses. The sample may be analysed directly for a given reaction product. Alternatively, where reagent has been applied to one sample in the array and has reacted with the one of the sample treated, it would be possible to analyse the array of samples and the detection of a reaction product would be assigned as being derived from the one sample treated.
The image can be recorded by any suitable means including recorded as an electronic or digital image. In one preferred form, the image is generated from a scan of the samples stained or illuminated to allow the samples to be visualised and the application of the reagent or reagents is carried out using a chemical printer application system where the reagent or reagents are discharged to the desired sample by the chemical printer.
It will be appreciated that steps (a) and (b) can be repeated or cycled so as to carry out a series of manipulations of the same sample or a number of different samples in the array. The multiple manipulations can be with the same reagent, the same set of reagents, or a number of different reagents.
The analysing is preferably by liquid chromatography, photoelectrical, photochemical, laser, radiochemical, or mass spectral analyses. It will be appreciated that the sample can be analysed directly for a given reaction product.
In order to generate an image of the samples in the array, it is usually necessary to make them identifiable in some manner. Labelling the samples with a visible marker is one example that would allow the visualisation of the position of the samples with a charged coupled device (CCD). A scan of the labelled samples would then be recorded digitally and stored in a computer for example. Once the image has been recorded in digital form for example, there would be no need to maintain the visualisation of the samples on the array as the image is maintained electronically. If the locations of the samples are recorded on an X/Y grid, this would be one way of accessing the positions of the samples electronically or digitally. The computer would also control the application of the reagent in step (b) to the position of the sample to be manipulated. The amount of chemical delivered to a sample would be regulated in the same manner as grey-scale for black and white printing. The position of all the samples would be known from their co-ordinates on the grid, for example, and so further manipulation is possible regardless of whether or not the samples are still visible.
The present invention is particularly suitable for the multiple analyses of one or more samples, particularly on an array like a protein blot. The method is applicable for N- and C-terminal determination of proteins and peptides derived from proteins separated by, for example, two dimensional gel electrophoresis. It will be appreciated that the method can be used for antibody or antigen assays of multiple samples and the like and the present method makes possible the different subsequent steps based on the outcome of an earlier reaction.
In one preferred form, the invention concerns the development of a chemical printer which sprays a chemical reagent to a sample which has been absorbed onto a solid support so as to cause a detectable reaction with the
Gooley Andrew Arthur
Packer Nicolle Hannah
Williams Keith Leslie
Hamilton Brook Smith & Reynolds P.C.
Proteome Systems Limited
Wachsman Hal
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