Patent Application
20060068396
The present invention relates to a method for a biochip; more particularly, relates to a method for a gene biochip operation technology platform on which the functions and the applications of the gene biochip can be effectively worked out and the practical applications of the gene biochip on related fields can be conclusively popularized.
A biochip is a micro device, comprising a silicon chip, a glass or a macromolecule as a substrate to integrate organic molecules (ex. nucleic acid or protein) by minimization technology to examine or analyze bio-molecules.
Because of the small size and the rapid reaction of the biochip together with its capability in parallel analysis of a great amount of biological information, the biochip can be used in biochemical treatment, biochemical analysis, biochemical examination, new medicine investigation and environment monitoring.
One of the most complete and eye-catching research now is the biochip research, characterized in that:
A. Examine a great amount of various genes at a time: According to the different goals of the researches, the researchers can choose different nucleotide fragments to fix on chip in arrays. In general, the researchers can investigate the actions of the genes in the cell with the nucleotide fragments for rapid and mass examination. It also changes the actuality of that a researcher usually did his research of one gene only in his whole life.
B. Require less biological material: The required specimen samples, bio-probes and targets are over 20 times lesser than that of the conventional “dot blotting.”
C. Automatic analysis: Because of the support of different software for related biological information, the analysis of the results from the gene biochip can be fully automatically done. Not only two basic functions of gene expression and differential gene searching can be achieved, but also multiple applications can be derived from the mutual exercises of the two functions. These functions and applications can be applied to many fields, such as biomedical science, food examination, chemical synthesis, new medicine researches, military applications, etc.
Yet, until now, the development of the gene biochip mainly focuses on two directories:
A. Glass chip: Nucleotide fragments can be fixed and arranged in arrays on a general glass chip in a special way to prepare required gene biochip. Such a chip can not be popularized to applications in related fields; and the reasons are as follows:
B. Nylon membrane chip: When preparing a biochip, the required nucleotide fragments are fixed on the nylon membrane. Comparing to the glass chip, a nylon membrane chip is easier to prepare; the threshold for the related experiment technology is lower; the experiment steps for the chemical calorimetric reaction is easier; and its reagent is cheaper. But, the required amount of specimen (ribonucleic acid) for the nylon membrane chip is larger and the analysis for the results of the chemical calorimetric reaction is not automatic so that the sensitivity and the accuracy are critically affected. Consequently, the nylon membrane chip is gradually replaced by the glass chip.
Besides, either the glass chip or the nylon membrane chip requires automatic spotting device. But the automatic spotting device costs quite high and is not easy to operate. Even those labs who can operate the device can not prepare the required chips independently which are prepared by some other biochemical companies. So, the technology for preparing the general gene biochip mentioned above does not fulfill the requirements of the user on actual applications.
Therefore, the main purpose of the present invention is to effectively work out the functions and the applications of a gene biochip by a method for a gene biochip operation technology platform which is with low cost, easy operation and high efficiency.
Another purpose of the present invention is to conclusively popularize the practical applications of gene biochip on related fields.
To achieve the above purposes, the present invention is a method for a biochip detecting limited cells, comprising the following steps:
A. Choose a nylon membrane chip having nucleotide fragments arranged in a dot matrix way by using a manual spotter.
B. Naturally dry the chip by heat after spotting; and fix the nucleotide fragments by a rapid nucleic acid cross-linker so that a biochip is prepared.
C. Collect some normal whole blood as a specimen.
D. Extract the ribonucleic acid in the whole blood to be linearly amplified.
E. Synthesize required amount of cDNAs (Complementary Deoxyribonucleic Acid) by a reverse transcription; and label the cDNAs to obtain a marker as a probe.
F. Process labeling, hybridization and post-hybridization to the chip and the marker.
G. Process chemical color reaction to the chip and the marker after the post-hybridization.
H. And, automatically analyze the result image after the chemical color reaction.
Accordingly, by the above steps, a method for a gene biochip operation technology platform with low cost, easy operation and high efficiency is obtained so that the functions and the applications of the gene biochip can be effectively worked out and the practical applications of the gene biochip on related fields can be conclusively popularized.
The present invention will be better understood from the following detailed description of preferred embodiments of the invention, taken in conjunction with the accompanying drawings, in which:
Table. 1 is a table showing the diagnostic results come from the technology platform of diagnostic chip for the nucleic acid in limited cells which is confirmed by Real-Time PCR (Polymerase Chain Reaction) according to the present invention.
The following descriptions of the preferred embodiments are provided to understand the features and the structures of the present invention.
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A. Obtain a nylon membrane chip having nucleotide fragments arranged in a dot matrix way by using a manual spotter, where each nucleotide fragment is obtained by soaking a nucleotide fragment with a di-distilled water to a size of 100 nl (nanoliter) with a length of 200 μM (micrometer) together with an interval of 1.5 mm (millimeter) between each two fragments. The manual spotter 1 comprises a base 11, a chip settlement layer 12 deposited on the base 11, a chip fixing layer 13 deposited on the chip settlement layer 12, and a dotting layer 14 deposited on the chip fixing layer 13. The chip settlement layer 12 and the dotting layer 14 are connected together at a side with a pivot 15 so that they can be opened at a proper position of the other side by an opening handle 141 of the dotting layer 14. The dotting layer 14 comprises at the center an area having a plurality of holes 142; the area is a square area of 4.5 cm (centimeter)×4.5 cm having 196 holes 142 (in 14 lines and 14 columns) each of whose inner diameter is 1.2 mm and is to be inserted with a trace suction tube (i.e. spotting nozzles) later.
The method for using the manual spotter comprises the following steps:
B. Naturally dry the chip by heat after spotting. And fix the nucleotide fragments on the nylon membrane chip by a rapid nucleic acid cross-linker so that a biochip is prepared. The nucleic acid cross-linker is processed with a crosslink in an energy of 1200 joule to fix the nucleotide fragments on the nylon membrane chip.
C. Collect some normal whole blood as a specimen. Only 1 c.c. is required to acquire 40 units of cDNAs (Complementary Deoxyribonucleic Acid) for detection; and only 4 units of cDNAs are required for each chip experiment.
D. Extract the RNA (ribonucleic acid) in the whole blood for linear amplification.
E. Synthesize required amount of cDNAs by a reverse transcription. And label the cDNAs to obtain a marker as a probe.
F. Process labeling, hybridization and post-hybridization to the chip and the marker. The time for labeling, hybriddization and post-hybridization is 24 hours.
G. Process chemical color reaction to the chip and the marker after the post-hybridization. During the post-hybridization, the time for detection is 30 minutes so that the calorimetric reaction in the post-hybridization is speeded up; and the color of the background will not be over-colorimetric and the false positive reaction can be reduced. The membrane washing which is before the blocking and the detection and is after the post-hybridization can be done with self-made washing buffer. And the membrane before the colorimetry can be processed by a self-made detection buffer.
H. Automatically analyze the result image by general density-analysis software after the chemical color reaction.
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Consequently, the present invention comprises the following characteristics:
A. The Development of a manual spotter: In the past, the automatic fabrication machine was too expansive to be popularized and not easy for operation; and its pump was not stable. The nylon membrane is apt to be contaminated so that the sizes of the spots made by the traditional dot blotting are not of the same size. And the traditional dot blotting can not produce many spots of nucleotide fragments on the same nylon membrane at a time. To solve the above problems, the present invention develops a manual spotter with easy operation as well as the capability of blotting many spots of genes at the same time, so that the genes can be blotted in a matrix way to a size of 100 nl with an interval of 1.5 mm between each two dots.
B. Biochip preparation: To popularize proper applications of the biochip on related fields, the present invention chooses the nylon membrane instead of the glass membrane to solve the problems of high cost and high technology threshold. After obtaining a nylon membrane, required amount of nucleotide fragments are spotted on the chip in a matrix way by the manual spotter according to the present invention. The fragments are made by being soaked with a di-distilled water to a size of 100 nl with a length of 200 μM (micrometer). After the manual spotting, the chip is dried with heat. And, by a rapid nucleic acid cross-linker, the nucleotide fragments are processed with a crosslink by an energy of 1200 joule to be fixed on the nylon membrane chip and so a biochip is prepared.
C. Specimen treatment: Up to now, the required amount of the nucleic acid for a biochip experiment is great. Take the most common used glass biochip microarray for example, the value of A260/A280 of the nucleic acid (spectrophotometric absorbance ratio at 260 nm by a 280 nm wavelength, while ‘nm’ means ‘nanometer’) is strictly required to be above 1.8 and the amount of the nucleic acid has to reach mRNA 2 μg (total RNA 100 μg, while ‘mRNA’ means ‘messenger RNA’ and ‘μg’ means ‘microgram’). Because the specimen is not easy to obtain, the practical applications of the biochip is limited to the examination of microorganism and organ abundant in RNA only; and so the popularization and the development of the biochip are held back too.
So, the present invention improves the traditional experiment method to a novel method of treatment for the specimen. The RNA is linearly amplified to solve the problem of the limitation of specimen and to stabilize the amount of required cDNA synthesized by reverse transcriptional test. Even with tiny amount of RNA in the specimen, the reaction of the biochip is still available. Not only is the range for the quality requirement broadened (only 1.2 of A260/A280 is required) but also only 1 c.c. of whole blood is required to obtain 40 units of cDNA and only 4 units of cDNA is required for each biochip experiment.
D. The simplification of the experiment steps for labeling, hybridization and post-hybridization: All experiment steps are easy and the equipments are simple according to the present invention that only a temperature control machine is required to finish all the steps. The time for labeling, hybridization and post-hybridization is prolonged to 24 hours which further solves the problem of weak reaction for tiny amount of specimen. Besides, the time for detection during post-hybridization is prolonged from 5˜10 minutes to 30 minutes so that the calorimetric reaction is speeded up; the background color will not be over-colorimetric; and the False Positive reaction can be reduced.
In addition, the required reagents for the blocking buffer, the washing buffer and the detection buffer can be self-made with the following ingredients: [1] Blocking buffer—50 grams of blocking reagent; [2] Washing buffer—0.4% SDS (Sodium Dodecyl Sulphate) or 0.5×SSC (Sodium Chloride Sodium Citrate); and [3] Detection buffer—0.1M (mole/molar) Tris-HCl, 0.1M NaCl, or 50 mM (milli-mole/molar) MgCl2●6H2O. And, the detection and the secondary antibody can be recycled. Therefore, common laboratories and related authorities can finish all the experiment steps independently.
E. Automatic analysis: The image acquired from the experiment can be automatically analyzed by density analysis software, which solves the problem of the inaccurate judgment to the experimental results of the calorimetric reaction in the past. The density analysis software can be Alpha Ease FC Stand Alone V.3.1.2 or GeneTAC™ Integrator Version 3.3. And the software is easy to operate and is low in cost. The results obtained can also be further utilized by related software of biological information according to the user's requirements.
To sum up, the method for a biochip detecting limited cells according to the present invention, not only surmounts the difficulty of popularizing the traditional chip experiment and make the gene biochip be able to be practically applied in related fields; but also practical applications of related researches or examinations can be run more smoothly, such as the analysis of the function and the behavior of a gene, the analysis of a gene mutation, the differential gene screening, the transcription factors searching, the gene study and invention of a new medicine, the clinical disease examination, the treatment follow-up, the food diagnostics, the agricultural product improvement and research, etc.
The preferred embodiments herein disclosed are not intended to unnecessarily limit the scope of the invention. Therefore, simple modifications or variations belonging to the equivalent of the scope of the claims and the instructions disclosed herein for a patent are all within the scope of the present invention.
