Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-27
2001-02-13
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S283100
Reexamination Certificate
active
06187537
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the creation of a DNA analyzing array by separating DNA into individual genes, replicating the same and solubilizing DNA genes in a solution of tens, hundreds or thousands of distinct microscopic squares called “features” on a gene chip or substrate.
BACKGROUND OF THE INVENTION
Typically, the DNA is separated into individual genes and replicated many times in a number of
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well plates (an industry standard) and minute pieces of DNA are positioned on an underlying substrate such as a chip from the DNA genes solubilized in a solution. After forming the DNA wet array and drying the DNA, the completed array is bathed in a solution of two or more fluoresces labeled total genomic tags, with the tags hybridizing to bind to a particular gene on the array by causing the fluoresces to fluoresce and by measuring the intensity of the signals, determinations may be made between the various features.
To date, the creation of such array is complicated, and while arrays have envisioned in terms of several thousand features per substrate area, such arrays are produced in terms of days rather than minutes. Further, while the well plates can store the individual genes within respective wells, over time the machine forming the wet arrays requires constant cleaning to deter contamination of the arrays. Once created, such gene chips are useful in testing for dozens of genetic diseases of different severity, and the test can be cheaply and quickly effected. Chips have been produced; however, significant energy is required to realize a practical chip.
Aeffymetrix, Inc. has recently disclosed an approach utilizing a glass slide as a substrate, about half the size of a postage stamp with thousands of distinct microscopic squares (features), each attesting for a specific DNA sequence. The features on the glass surface are covered with a compound containing chemical protecting groups that block further chemical reaction. Optically, collected protecting groups can be removed. A thin mask is then pulled over the chip containing holes to allow light to strike specific features, with the other features on the chip remaining protected. Subsequently, the chip is washed with a solution containing one of four DNA components called nucleotides (A, C, G or T). The DNA component washed solution binds only to the unprotected features. Each incoming nucleotide carries its own protecting group so that the washed features are reprotected. Sequentially, a new mask with different pattern of holes and optical (light) impingement removes the protecting groups at the different pattern of holes associated with a second group of features. In a multi-cycle process, chains of precisely ordered nucleotides are built onto each feature.
As may be appreciated, genes are made of two strands of DNA nucleotides of a specific order, bound to each other like the halves of a zipper. Nucleotide binding is governed by certain relationships. For instance, the nucleotide T always binds with that of A, but never with C or G, or with another T. Thus, a strand of nucleotides has a single complimentary strand which will match it and bind exactly. Thus, a chip (or other substrate) containing nucleotide strands of a given composition can find specific mutations in a person's genes. Man has approximately 80,000 genes. Therefore, a DNA gene array of closely spaced features or dots of microscopic size may be constituted by as many as 400,000 dots on a single substrate and capable of carrying all DNA's for several persons, or one person in redundancy.
In the production of the liquid DNA gene arrays, DNA is extracted typically from tissue cells grown in cultures, the DNA is fragmented into thousands of pieces which can be chemically labeled with a fluorescent compound. The pieces contain parts of genes or whole genes. Thus, each feature of a chip contains a nucleotide strand of a normal or mutant section of a known gene. Thus, all possible mutations of a gene can be detected by features on a single chip and all may be tested simultaneously. By use of an optical scanner, the features on the chip can be read for fluorescent color and intensity. Features containing fluorescently labeled DNA may provide signals fed to a computer as input data, with that data being analyzed to provide information as to whether the person providing the genes carries one or more mutations, and further the identification of the mutation itself.
It is therefore a primary object of the present invention to provide a high throughput test system and components for ascertaining genetic mutations enhanced by the dry, orderly world of computer hardware in contrast to the wet and messy world of living tissue and of liquid DNA gene features applied to the slide by effecting a dry DNA transfer film to create in turn, a dry DNA analyzing array of features or spots on such slide.
SUMMARY OF THE INVENTION
The present invention, in one form, is directed in part to an improved high throughput process of forming a dry DNA transfer media, such as film, paper, nitrocellulose, plastic or glass. For example, a thin flexible, resilient film sized to the top surface of a generally rigid well plate having within such upper surface a plurality of closely spaced wells in column and line fashion within which are pre-placed separated, replicated DNA genes solubilized in a solution. The roughened surface of the thin flexible resilient film is sealed to the upper surface of the well plate. Means are provided for effecting a rigid film plate assembly. The assembly may then be inverted to cause the DNA solution under gravity to physically, locally wet coat the roughened surface of the film, with the roughened surface causing the DNA gene solution to cling to the film while preventing the DNA gene solution from running radially from one spot to another. The assembly is then reinverted to its initial position, and the DNA gene solution spotted film is removed slowly from the well plate. Upon drying, the DNA gene solution spot coatings thereon form a dry DNA transfer film capable of physical and chemical dry transfer of DNA to a substrate.
The spot diameter or dimensions of the same and the spot configuration depend on the size and configuration of the wells within the well plate. The DNA gene solution spots may be air dried to speed the process. A vacuum seal may be effected between the thin flexible resilient film and the underlying well plate to momentarily fix the film to the well plate prior to and while inverting the assembly. The thin flexible resilient film and well plate assembly may be secured in a fixture or jig to facilitate rendering the assembly components fixed during the inverting and reinverting steps and to maintain the seal between well plate and film during the initial liquid coating of the roughened surface of the film and to prevent the DNA gene solution from running between the wells.
In another aspect, the present invention involves a dry DNA transfer film as a product by the process described above.
In a further aspect of the invention, a dry form DNA analyzing process includes the following steps:
(a) sealing a flexible resilient film to the upper surface of a rigid well plate having a plurality of spaced wells opening to the upper surface and facing the film, the wells being prefilled with respective, separated, replicated DNA genes solubilized in a solution;
(b) forming a fixed, sealed assembly between the well plate and the overlying thin flexible film;
(c) inverting the assembly to transfer DNA gene solution spots to the facing surface of the film over localized areas of said film defined by respective wells;
(d) reinverting the assembly, removing the film and drying the transferred DNA gene solution spots to thereby form a dry DNA gene transfer film;
(e) placing the dry DNA gene transfer film in a position facing a flat glass test substrate and applying force and movement such as momentarily impacting the face of the DNA film on a face opposite that bearing the spots at the spot locations to cause dry DNA to forcibly
Francart, Jr. Armand
Zinn, Jr. Donald E.
Houtteman Scott W.
Sughrue Mion Zinn Macpeak & Seas, PLLC
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