The Establishment And Preliminary Application Of HLA-B Oligonucleotide Typing Chip

Oligonucleotide chip has developed rapidly and its method is diverse according to the purpose. The surface treatment of the chips is one of the key techniques. By our own method we prepare oligonucleotide chips with 3-Aminopropyltriethoxysi-lane as the surface treatment reagent and design the typing probes for the HLA-B antigen genotyping.At first the chips were rinsed in the lotion by the ultrasonic, then were dealt by 100 mmol/L NaOH to generate free hydroxyl on surface, and were embellished by the 3-Aminopropyltriethoxysilane as the bifunction reagent. In the end the embellished chips could integrate amidocyanogen with covalent bond. After the amidocyanogen embellishment, the probes were blotted regularly on the chips according to predesigned order. The blotted chips could be hybridized after hydrating, rinsing, roasting and riveting.The oligonucleotide chip technology displays the predominance by the celerity, high flux and tremendous information detection. Human leukocyte Antigen (HLA) system is one of the most important genic systems of regulating special immune response of human and deciding the individual difference for disease susceptibility and hundreds of diseases have been found to relate with HLA-B, especially the diseases activated by the hierarchy and environment, so it is very significant to detect the typing of HLA in scientific research and clinical medicine practice. Due to the numerous alleles and multitudinous polymorphism of HLA, there are estimate error in the typing of HLA by traditional serology and cytology methods, especially in HLA I typing. According to the common HLA-B alleles and the characteristic of clinical sort discernability in Northern Chinese, and considering the hereditarydisease related with the HLA, such as Ankylosing Spondylitis and Infancy Diabetes, we designed 66 specific typing oligonucleotide probes with medium distinguishability.We extract genomic DNA from human blood cells by TKM method as the template, label primer with cyanine(Cy3), confirm the proportion of two primers, amplify HLA-B gene by unsymmetrical PCR, scan signals from hybridization of PCR product and probes by ScanArray apparatus, analyze the positive probes using correlative computer software and obtain the results. With these probes, we certified 19 HLA-B alleles from HLA-B7 to HLA-B83, contrast polymerase chain reaction with sequence specific primers (PCR-SSP) genotyping method with this method, the gene chip could attain the medium distinguishable purpose. HLA-B genie chip has considerable precision and specificity. It could check out the multitudinous samples in one chip and apply to the clinical HLA-B typing.