Gene chip technology progress and its applications (1)

1 gene chip overview

As the human genome project (Human Genome Project) that is full of nucleotide sequencing is nearing completion, human genome research focus gradually entered the post-genomic era (Postgenome Era) to gene functions and gene diversity.

Through the individual at different growth stages or different physiological state, a large number of parallel analysis of gene expression, research the corresponding gene in vivo function, clarify the different levels of multiple gene synergy mechanism resulting in human disease such as cancer, cardiovascular disease pathogenesis, diagnosis, treatment, drug development and research play a huge role. It will significantly promote human genome structure and function of the genome of the various genomics projects.

Gene Chip works and classics of nucleic acid hybridization methods (southern, northern) is the same, and all application known as probes and nucleic acid sequence complementary targets nucleotide sequences hybridized, through subsequent signal detection for qualitative and quantitative analysis, gene chip in a tiny substrate (glass, plastic, silicon wafers, Tablet, etc) have a large number of surface integrated molecular recognition probe, at the same time a parallel analysis of a large number of genes, to large volumes of screening and detection analysis.

Gene chip technology processes including: chip design and preparation; mark target genes; chip hybrid and hybrid signal detection.

Gene chip design actually refers to the chip on the choice of nucleic acid probe sequences and arrangement, and design methods depends on its application, the current application including gene expression and transcription of analysis and target sequence single base polymorphic loci (single nucleoTIde polymorphism, SNP) or mutation points detection, expression-chip is in hybrid experiments on multiple different State samples (different organizations or different developmental stages, different drugs stimulate) thousands of gene expression differences for quantitative detection probe sequences generally comes from a known gene of cDNA or EST libraries, design time sequence specificity should be placed in the first place, to ensure that test target gene of specific binding, for the same purpose gene can design multiple sequences do not duplicate the probe, the final data more reliable.

Gene polymorphisms on single base detection of chips generally use length shift design, i.e. target sequence from beginning to end, with a length of complementary nucleotides sequence to form a probe, which set tip is and exactly matches the target sequence of wild-type probe, and then for each of the wild type probe, which position between a base with other three bases replacement, creating three different single base variations of nucleotide probe, this design allows for a certain period of nucleic acid sequence all possible SNPs loci for scanning.

Chip fabrication method consists primarily of two types: (1) point-like law: first, probe preparation, on the basis of gene chip analysis of target gene from the relevant database to select the specific sequence PCR amplification or direct synthetic oligonucleotide sequence, and then by computer-controlled three-coordinate work platform using special needles and micro-sprinkler respectively different probe solution by point allocation in glass, nylon and other solid substrate of different sites, through physical and chemical methods to fixed, the method the technical links are more mature, and flexibility, suitable for research units wanted preparation dot matrix gene chip-sized.

(2) in-situ synthesis method: it is in the glass, and other hard surfaces directly array of synthetic oligonucleotide probes, the current application of the main light to protect the parallel synthesis, synthesis of piezoelectric print etc, the key is the high spatial resolution of template location technology and high-yield of DNA synthesis chemical synthesis technology, suitable for the production of large-scale DNA probe chip to achieve standardization and high-density chip-scale production.

Pending the preparation of the analysis sample microarray experiment process is an important link, target genes in combination with chip required before you probe hybridization for separation, amplification and tags.

Tag method according to the sample source, chip types and research purposes. Usually in the test sample of PCR, RT or in vitro transcription process realization on target gene marker. For detection of intracellular mRNA level chip, generally required from cells and tissues for RNA, RT, and join coupling with markers dNTP, thereby complete the target genes of the labeling process, for array density smaller chip can use isotope, the instruments are laboratory general use device, easy to carry out related work, but the signal detection, signal stronger certain hybrid raster prone Halo around the analysis of signal interference. High-density microarray analysis generally use the FITC target gene, the settings through appropriate confidential and the fluorescence signal strength of the standardization of the cellular mRNA for quantitative detection. In recent years, the use of multicolor fluorescent markers can be more intuitive for you to compare different source samples of differential gene expression, that the target genes from different sources with different excitation wavelength of FITC, and at the same time and the microarray hybridization, by comparing the chip on the distribution of different wavelength fluorescence for different sample graphs differentially expressed genes in Atlas, a common two-color fluorescence reagents with Cy3 and Cy5--dNTP dNTP. On polymorphism and mutation detection-gene chip with multicolor fluorescence technology can significantly improve the accuracy of the chip and detection range, for example with different fluorescein individually mark target sequence and single bases mismatch of the reference sequence, make them at the same time and chip hybrid, through different fluorescent intensity of that target sequence of bases mismatch message.

Gene Chip and target genes of hybrid process and general molecular hybridization procedures are basically the same, hybridization reaction conditions according to the length of the probe, GC base type of the content and the tablets to optimize, as for detection of gene expression, the strictness of hybrid less for mutations of the chip temperature high of hybrid, hybrid conditions relatively short time.

If you are using isotope labeled target genes, subsequent signal detection that is autoradiography, with fluorescent markers,Need a fluorescence scanning and analysis system, the corresponding probe array conducting analysis of fluorescence intensity, the resulting test samples of the corresponding information. Due to the amount of gene chip gets big, microarray hybridization data analysis, processing, query, compare, and so need a standard data format, at present, a large database of gene chip is built, you will get various laboratory microarray results together in order to facilitate the exchange of data and the results of the assessment and analysis.

2 application of gene chip

Gene expression of a gene chip to draw is currently the most widely used areas of the human genome project, an important part, which provides an overall analysis of cell expression of information, but also to understand and some special life phenomena related gene expression provides a powerful tool for gene and gene interaction mechanism.

Human genome coding about 100000 different genes, therefore, have a large number of mRNA surveillance tools is important. Gene chip technology can clearly directly and quickly detect to level 1: 300000 mRNA that appears, and easy-to-thousands of genes at the same time monitoring. At present, has to be able to synthesize 1.6cm2 area and read the included 400000 probe arrays, you can monitor 10000 gene expression. Stanford Brown preparation of yeast using cDNA microarray for yeast in different cell cycle status as well as heat shock cold shock treatment after its 2473-gene expression Atlas, a visually reflects the different conditions and State regulation of gene transcription, thus to find gene regulation mechanism provides an effective way.