Genetic chip technical progress and application
A genetic chip summary
With the Human Genome Project (Human Genome Project) Namely all nucleotide check order namely completion, the intersection of weight and genome era (Postgenome Era) enter gradually, that human genome study To the function of the gene and diversity slope of gene. Through to individual in grow a large amount of parallel analysis of gene expression under the developmental stage or different physiological state while being different, study the corresponding gene in the inner function of organism, expound the mechanism of the multigenic synergism of the different levels, in human great disease such as cancer, pathogenesis, diagnose treatment, the intersection of medicine and the intersection of person who develop and to study, play an enormous role respect, cardiovascular vessel of disease. It will promote every genome of human structural genome and function genome to study the plan greatly.
The operating principle of the genetic chip hydridizes the method (southern, northern) with classical nucleic acid molecule It is concerted, employ known nucleic acid arrays to hydridize with complementary target nucleotide sequence as the probe, the signal detection through being subsequent determines the nature and quantitative analysis, the genetic chip is in the chip (silicon dice, glass slide, plastic sheet,etc.) of a minification It is last probe molecule a large number of not to integrate surface,can analysis a large number of genes parallel at the same time, carry on screening and detection analysis of the large information volume. The main technological procedure of the genetic chip includes: Design and preparation of the chip; Mark of the target gene; The chip hybridizes and hybridizes signal detection.
Gradate choice of the probe that the design of the genetic chip refers to nucleic acid on the chip in fact and arranging, the design method depends on its purpose of application, the present application area mainly includes gene expression and duplicates some (single nucleoTIde polymorphism of polymorphism location of single base in atlas analysis and target array, SNP) Or the detection of catastrophe point, the purpose of the expressing chip is a plurality of different state samples (different organizations or different developmental stages in hybridizing the experiment, stimulate with the medicine) The expression difference between thousands of genes of China is carried on quantitatively and measured, the probe array generally comes from cDNA or EST storehouse of the known gene, the gradate specificity should place on primary position while designing, in order to guarantee the peculiar combination with the purpose gene to be measured, can design a plurality of the intersection of array and repetitional probe as to the same the intersection of purpose and gene, make the final data more reliable. Genetic the intersection of form and the intersection of base and chip that polymorphism measure the intersection of person who adopt and long shift design the law generally, namely form a probe of associations according to the complementary nucleotide sequence that the target array fetches certain length sequentially from the beginning to the end, this group probe the intersection of target and the intersection of array and the intersection of wild type and probe of perfect matching, to every wild type probe, a certain base in the position will be replaced with three kinds of other bases separately among them, form the nucleotide probe that three kinds of different single bases change, this kind of design can click and scan some segment nucleic acid array all possible SNPs location.
The chip preparation method mainly includes two kinds of types: (1)The law of sample application: It is the preparation of the probe storehouse at first, choose the peculiar array to carry on PCR amplifying or widowed nucleotide sequence of direct artificial synthesis from the associated genetic database according to the analysis goal of the genetic chip, then assign different probe solutions in the glass piecemeal respectively with special syringe needles and little shower nozzles through three computer-controlled coordinate working platforms, at nylon and other the intersection of solid phase and the intersection of chip and external imparity order, enable fixing it through physics and chemical method, every technological link of this method is relatively ripe, and the flexibility is big, suitable for the research unit and prepare the M moderate genetic chip of scale of dot matrix by oneself depending upon need. (2)The synthesis method of normal position: This law is a synthetic oligonucleotide on hard surfaces such as the glass,etc. directly probe array, the applied main glazed synthesis method of deprotecting running side by side at present, prints synthesis method,etc. on the piezoelectricity, its key is a high spatial resolution template location technique and high DNA chemical synthetic technology of formating the productive rate, suitable for producing the large-scale DNA probe chip, realize canonicalization and large-scale production of the dense chip.
An important link that the preparation which treats analytical samples is the experiment procedure of the genetic chip, the target gene must be separated for before binding with chip probe and hybridizing, amplifies and the mark. The labeling method is different to some extent on the basis of difference between sample source, chip type and research purpose. Usually realize the mark to the target gene while PCR of the sample to be measured amplifies, goes against and duplicates or duplicates in vitro. MRNA expresses the lateral chip for measuring in cells, generally need to draw RNA from cells and the tissue, go against and duplicate, and add coupling dNTP with the label, thus finish the mark course to the target gene, as to the thing that the minor chip of array density can use the isotope, the required instrument is that the laboratory uses the apparatus conventionally, easy to launch the relevant work, but during signal detection, the dot matrix of some hybridization with strong signal is prone to the halation, interfere with the analysis of the signal around. The analysis of the dense chip generally adopts the fluorescein mark target gene, and can carry on the expression of mRNA in cells quantitatively and measure to the mark of fluorogenic signal intensity through the arrangement of the appropriate confidential reference items. Gene expression difference of different source samples more ocularly of polychromed fluorogenic mark technology used in recent years, different target gene of source with different fluorescein mark to stimulate wavelength, and make them hydridize with genetic chip at the same time, through comparing the fluorescent distribution map of different wavelength on the chip obtains atlas that the difference expressed the gene between different samples, commonly used double-colored fluorogenic reagent is Cy3- dNTP and Cy5- dNTP. Adopt reliability and detection range that polychromed fluorogenic technology can improve the chip to polymorphism and mutation measuring genetic chip greatly, for example differentiate the mark target array and reference array of the single base mismatching with different fluoresceins, make them hydridize with chip at the same time, produce information of base mismatching in the target array through the comparison of different fluorogenic power.
It is basically the same that the genetic chip hydridizes the course with hybridization course and general molecule of the target gene, hybridizing the condition reflected will optimize according to the length of the probe, type of GC base content and scene, measure such as being used in gene expression, the strictness hybridized is lower, and the hybridization of chip used for measuring in sudden change is tall in temperature, hybridize time short, the condition is relatively strict. If use the isotope mark target gene, following signal detection is radiating developing, if use the fluorogenic mark, need a set of fluoro scanning and real-time analysis system, is analyzed and compares the fluorogenic intensity on the corresponding probe array, thus get the corresponding information of the sample to be measured. Because the information volume that the genetic chip is obtained is large, hybridize machine format analysis, treatment, inquiry, comparison,etc. to need a canonial data format as to the genetic chip, at present, one bull database of genetic chip in course of constructing, concentrate to stand up every laboratory acquired result of genetic chip, in order to benefit the machine format exchange, consequential assessment and analysis.
Application of 2 genetic chips
The drawing of the atlas of gene expression is that the genetic chip employs the most extensive field at present, also the important constituent element of the human genome project, it has provided information on analyzing cells express the state on the whole, and offered the strong tool for understanding the gene expression correlated to some special biological phenomenas, adjust and control as to the gene and the discussion of gene interaction mechanism is of great importance. About 100000 different genes of human genome code, so, experiment tool which monitors a large number of mRNA is very important. The genetic chip technology can be detected by 1 fast directly plain: MRNA that 300000 levels appear, and easy to monitor hundreds and thousands of genes at the same time. At present, can already formate and read the array containing 400000 probes on 1.6cm2 area, can monitor the expression states of 10000 genes. Brown of Stanford University uses yeast cDNA chip prepared, obtain yeasts in different cell cycle state and expression atlas of their 2473 genes after hot shock and cold shock are dealt with, the gene duplicates and adjusts and controls competence after reacting under different conditions and state more ocularly, thus has offered a valid route for the mechanism that looked for the gene to adjust and control.















