| background and objectHuman leukocyte Antigen is the most complicated and polymorphism heredity system that has been discovered up to the present. The 13th International Histocompatibility Workshop and Conference (IHWC) , which was held in February 2001, defined that the number of the discovered HLA alleles has reached 1340. The HLA system plays an very important role in antigen identification and presentation, immune response and regulation, and is also one of the key factors that affect the result of organic transplantation and hematopoietic stem cell transplantation. Most of the clinical types HLA by serology at the present time. But the extensive cross - reactivity and the lacks of the ideal antisera make it difficult in technique and the false result. Furthermore, the lack of the standard antisera also cause the " blank" result in a few newly discovered alleles. Labeled by the 12th IHWC held in 1996, HLA typing techniques enter DNA stage in general. For DNA typing in the single locus, PCR - SSP and PCR - SSOP were used more often now in the world. Although their result is more accurate than serology, the operation is too elaborate to use in large sample test. Gene-chip is a newly developed menthod which characterized as high - throughput and intensive, whose superiosity fits the tedious research of HLA system. Though there are a large number of reports in domenstic and abroad about this, no ma-lure HLA genotyping chip was produced until now . Id like to fabricate the HLA genotyping chip by the method developed in our lab, and typing HLA allele in DQA1. I am in an attempt to establish a mature technique process to typing the complicated HLA system.Materials and methods1. prepare samples extract human genome DNA from anticoagulatedwhole blood by phenol/chloroform method, exam the concentration and purity of DNA2. PCR amplification of the target sequence design a pair of primers ac-coding to the conservative region of HLA - DQA1 exon 2, and lable the sence strand by FITC. Make the fiuorescenced strand the advantage strand by assym-metric PCR.3. prepare the oligonucleotide probes designed 16 probes as four groups according to HLA - DQA1 sequence in GenBank and modify each probes by ammonia at 5'-end. Then resuspended the lyophilized probes in PH =9,0. 1 carbonate buffer to 100 M as explored in advance.4. prepare the spotting slides soak the cover slides in chromic acid solution for more than 12hrs, wash to clean, soak in NaOH, in acetone, in arm molecule solution for connecting, roast to fix the groups, ready for use.5. prepare the oligonucleotide microarray add 16 prepared probes into 384 -pole plate, meantime,add P2 ,low homologic probes and probe buffer in the same concentration as positive control , blank control and negative control respectively. Spot microarray as designed by the spotting machine. Then hydrate and roast' to fix the probes, ready for use6. block and hybridizate block and wash the prepared slides by the method developed in our lab. Mix the PCR products and the hybridization buffer, cover each microarray, preserve in the capsule, 55 C ,30 min to hybridize.7. wash and exam flush the cover slide, wash the microarray by 1 SSC/ 0. 1 % SDS, 0.5 SSC/0. 1 % SDS,0. 1 SSC and ddH2O which was prehea-tod lo 60 C resoectivelv. blow to dry. Exam and analyze the hybridization signalby Laser Cofocal Scantier and CCD imaging software. Determine the allele typeoi the sample.Result1. PCR amplification of HLA - DQA1Use the extracted genome DNA as template, amplify the target sequence. Examine the products by PAGE. The bands is clear and the length is 538bp as itshows,which matches the design.2. the optimize of the oligonucleotide probes preparationThe optimal condition is to resuspend the probes in PH =9,0. 1 M carbonate buffer to 100 M.3. the accuracy and stability of the typing resultTyping results match the SET results, and in 5 hybridization, the repeated rate is 91.2%. |
