Human leukocyte antigen HLA-A and HLA-B gene full-length sequencing method and HLA gene sequencing and typing method
A technology of HLA-A and leukocyte antigen, which is applied in the field of full-length sequence determination of -B, HLA gene sequencing and typing, and tissue matching related gene HLA-A, which can solve ambiguity and other problems.
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Embodiment 1
[0176] This example provides a complete set of schemes for obtaining 38 HLA-A 4.3kb full-length alleles and 30 HLA-B 3.7kb full-length sequences.
[0177] First, 45 samples containing common alleles in all sublines of HLA-A were selected from 1000 blood samples of Shenzhen hematopoietic stem cell donors who had undergone high-resolution sequencing of HLA-A and -B genes, and the results were known. 38 samples containing common alleles in all sublines of HLA-B were subjected to the gene sequence determination of the present invention. Take HLA-A gene full-length amplification primers A-Genome-F and A-Genome-R in the present invention, carry out HLA-A gene full-length amplification to 45 samples, take HLA-B gene full-length amplification primer For B-Genome-F and B-Genome-R, the full-length HLA-B gene was amplified on 38 samples, and the amplification was carried out in ABI 9700 PCR instrument. The amplification reaction system consisted of:
[0178] 10×Pfu Buffer 2.5μL
[0179...
Embodiment 2
[0195] This example gives an example of performing HLA-A and -B high-resolution sequencing typing on 384 cases of voluntary hematopoietic stem cell donor samples in Shenzhen.
[0196] The applicant randomly selected 384 samples with known HLA-A, -B allele types from hematopoietic stem cell voluntary donors in Shenzhen, and used the PCR primers and sequencing primers of the present invention to perform sequencing typing to verify the present invention implementation effect. Using the amplification and sequencing primers of the present invention to perform sequencing and typing experiments on the above samples, clear HLA-A and -B genotypes were finally obtained and the ambiguous results in the selected samples could be resolved.
[0197] Firstly, two pairs of PCR amplification primers are used to perform PCR amplification reaction on HLA-A and -B of the sample respectively, wherein, the first pair of amplification primers amplifies the first exon to the fourth exon of the two ge...
Embodiment 3
[0235] In order to verify the effectiveness of the HLA-DRB1 group-specific primers, in this example, 8 samples containing 8 group-specific alleles ( DR01, DR02 / 15 / 16, DR03 / 11 / 13 / 14, DR04, DR07, DR08 / 12, DR09, DR10) specimens, respectively with the 5' end group specific Primers and 3'DR-generic amplify the sequence from exon 2 to exon 3 of DRB1. The amplification reaction system used is:
[0236] 10×Buffer 12.5μL
[0237] MgCl 2 (25mM) 1.5μL
[0238] dNTP (25mM) 2.0μL
[0239] 1 μL each of PCR primers (10 μM )
[0240] Genomic DNA 100ng
[0241] Clontech LA enzyme (5U / μL) 0.5μL
[0242] ddH 2 0 to 25 μL.
[0243] The amplification reaction procedure used was:
[0244] 95℃ 2min
[0245] 95℃ 15Sec
[0246] 65℃ 15Sec
[0247] 72℃ 2min, 5 cycles
[0248] 95℃ 15Sec
[0249] 60℃ 15Sec
[0250] 72℃ 2min, 26 cycles
[0251] 95℃ 5Sec
[0252] 55℃ 1min
[0253] 72°C 3min, 4 cycles
[0254] 72°C 15min
[0255] 4°C Infinite.
[0256] Take 5 μL of the PCR product, stain...
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