Capillary array electrophoresis scanner

Details Capillary array electrophoresis scanner Capillary array electrophoresis scanner

1999-01-27

2001-08-07

Warden, Jill (Department: 1744)

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

C204S600000, C204S601000

Reexamination Certificate

active

06270644

ABSTRACT:

BACKGROUND OF THE INVENTION
Research leading to portions of the present invention was funded in part through the National Institute of Standards and Technology ATP Grant to Affymetrix, Inc.
AREA OF THE ART
The present invention relates to methods and apparatus useful for facilitating the sizing of biomolecules with electrophoresis. In particular, the present invention relates to high volume analysis of aliquots of solutions especially useful in the context of systems for electrophoretic analysis, such as Capillary Array Electrophoresis (“CAE”) of greater than 1000 capillary electrophoretic separations in parallel.
Analysis of Genomic DNA
An essential goal of biomedical research in the 21
st
century will be the complete analysis of genomic DNA—the blueprint for life. This effort will involve not only the human genome—the genome of many organisms, both plant and animal, will be of profound importance to the human community. The ever-growing drive to determine the DNA sequence of complete genomes has created the need for the integration of automated instrumentation, bioinformatics, and sequencing chemistries into a high-capacity, high-accuracy process. Besides the human genome, the complete genomes of other animals, bacteria, fungi, and plants will be sequenced in the not-too-distant future. In the next decade, literally tens of billions of bases of novel DNA sequence will be acquired. Along with those novel sequences, confirmatory sequencing and mutant analysis projects for known genes will push further for increased capacity and reliability of sequencing sample preparation and reaction setup. Simplification and full automation of laboratory processes will have to provide the necessary increase in throughput and capacity, while guaranteeing reliability and reproducibility, if these goals are to be met.
As just one example of the wide-reaching importance of this work, the discovery of new medicines has been revolutionized by genomics. The human genome is believed to contain about 100,000 genes. The full human genome will be sequenced by 2003 resulting in an explosive increase in the number of drug targets, which currently number about 500. An estimated 3000-10,000 potential targets will be identified by 2003.
Another strategy is called positional cloning, in which genes associated with familial diseases are sequenced, have been used in an effort to identify new drug targets. For example, specific genes associated with diseases such as obesity or schizophrenia open the possibility of treating such conditions with drugs acting on specific targets.
Still a third strategy for identifying the specific genes responsible for regulating cellular processes such as differentiation and neoplasia has resulted in new targets for drug intervention. An elegant and conceptually simple procedure called Differential Display has revolutionized the approach to this scientific problem. The principle of this procedure is to represent each RNA “message” from a tissue on a gel, thereby creating an “RNA fingerprint” for each sample. The banding pattern obtained from, for example, healthy tissue, can then be compared with the banding pattern from cancerous tissue. Bands present in one sample, but not the other, represent genes expressed specifically in that tissue—in this case the cancerous tissue. The genes can then lead to the discovery of new targets for drug intervention.
All of these new analyses require high-throughput, high-capacity methods. In the past, access to genetic information has been limited by the low throughput techniques and instruments available for nucleic acid analysis. High throughput methods have the potential to dramatically change this situation, but even further improvements are needed. Thus, it is clear that the achievement of these important new goals is dependent on new types of biological instrumentation offering high-capacity, high-accuracy data acquisition coupled to computer tools for analyzing the resulting data.
Genes consist of four different chemical subunits, or bases-adenine (A), guanine (G), cytosine (C), and thymine (T)—attached to a sugar-phosphate backbone. The order of these bases, for example, GATTACA, determines the genetic message that leads to the production of particular proteins by the cell. Thus, we may compare the information contained in the sequence of A, G, C, and T in a gene as similar to the information contained in the sequence of the individual letters of the alphabet in a word. It is clear, therefore, that determining this sequence in a gene is crucially important to understanding the function of the gene.
Older methods for sequencing genes were developed around 1977-1980. They involved the use of specific chemical reactions on the genes, and the use of radioisotopes to identify the sequence of the different bases. An important separation step in the older methods involved the use of gel electrophoresis to sort gene fragments by size. In this method, a gel containing an appropriate buffer solution is cast as a thin slab between glass plates. Each end of the slab is electrified by the application of electrodes, to produce a positive end and a negative end of the slab. A small amount of the sample to be analyzed is pipetted onto the slab and the constituents of the sample are allowed to migrate along the slab under the influence of the electric field. The position of the different constituents of the sample on the gel is then determined by their molecular weight. A single slab can be divided into several lanes to make possible the analysis of several samples at the same time.
These older methods were effective, but slow and expensive. They made possible the sequencing of 3000-10,000 bases per person year, at a cost of $ 1-5 per base. However, there are billions of bases in the human genome alone, and it is clear that the development of faster, high-capacity, high-throughput automated methods would be of crucial importance to achieve the goal of sequencing entire genomes.
Automated DNA Analysis
In 1986, a method was developed at Cal Tech that used colored dyes as tags rather than radioactive isotopes for the four bases. Smith et al., 1986, 321
Nature
674-979. Each of the four colors-green, yellow-green, orange-red, and red—corresponds to a different base, so that it is possible to identify the different bases by means of their colored tags. Not only did this eliminate the need for radioactive isotopes, which pose problems in safe handling and disposal, but it also made possible the use of automated equipment in conducting the analysis.
A key to automating these methods was the development of capillary electrophoresis (CE), to replace the older gel slabs. In capillary electrophoresis, the gel is contained within a capillary tube rather than being layered as a slab on a glass plate. The capillary tube is a narrow-bore structure for performing high efficiency separations. High electric fields can be applied along the capillaries without significant temperature increases, and since the electrophoretic velocity of the charged species is proportional to the applied field, CE can achieve rapid, high-resolution separation. CE thus offers the advantages of nanoliter injection volumes, exceptional resolving power, fast separations in times ranging from a few minutes to less than one hour, less heat production, higher voltages, and reduced sample preparation. J.P., HANDBOOK OF CAPILLARY ELECTROPHORESIS, CRC Press, Boca Raton 1994.
In the automated system developed at Cal Tech, genetic material is chemically chopped into smaller segments wherein the terminal base in each segment is identified by a colored tag. All of the segments can then be separated by the process of electrophoresis, which sorts the segments according to their size as they pass through the gel. As the segments reach the end of the gel in the order of their size, they are illuminated by a laser, which causes them to fluoresce in their characteristic color. The fluorescence is then read out to identify the size and base terminus of the segment. Swerdlow, H. & Gestland, R., 1990, 18
Nuc. A

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