Primer group, kit and method for amplification of human leukocyte antigen-B (HLA)-B gene, and primer group, kit and method for genotyping of HLA-B gene
An HLA-B, gene amplification technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of high DNA template integrity, full-length fragment length, and high cost
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Embodiment 1
[0094] Embodiment 1 Amplification of HLA-B gene
[0095] 1. Sample DNA extraction
[0096] DNA was extracted from 96 blood samples with known HLA-B genotypes using the QlAamp Blood Extraction Kit (QIAGEN). The concentration of the extracted DNA sample is determined by using an ultraviolet spectrophotometer, and the concentration of the extracted DNA sample is adjusted to 20-50 ng / μl.
[0097] 2. Design HLA-B gene amplification primers
[0098] According to the latest HLA-B gene sequence in the IMGT / HLA database (http: / / www.ebi.ac.uk / imgt / hla / ), such as figure 1 As shown, the HLA-B gene has 8 exons, multiple groups (two for each group) of suitable conserved regions are searched, and each group of conserved regions is guaranteed to cover the 8 exons of the HLA-B gene. In the found multiple groups of conserved regions, design appropriate amplification primers respectively. When several allele sequences are inconsistent, use degenerate bases, or design a new allele group-specif...
Embodiment 2
[0110] Embodiment 2 Amplification of HLA-B gene
[0111] This example is basically the same as Example 1, the only difference is that the first set of amplification primers is a sequence of less than or equal to 8 nucleotides added to the 5' end and / or 3' end of B-F1 and B-R1. In this embodiment, 8 nucleotides are added to the 5' end and 3' end of B-F1 and B-R1 respectively, and the nucleotides can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine), in this embodiment, 5'→3' at the 5' end increases GGCTACAT, and 5'→3' at the 3' end increases TTTGAACC, and the first set of amplification primers can be used to amplify Exon 1-3 of the HLA-B gene, the second set of amplification primers is the 5' end and / or 3' end of B-F2 and B-R2 to increase the sequence of less than or equal to 8 nucleotides, in this In the example, one nucleotide sequence is added at the 5' end and 3' end of B-F2 and B-R2 respectively. In this embodiment, 5'→3' at the 5' end increases G, an...
Embodiment 3
[0112] Embodiment 3 Amplification of HLA-B gene
[0113] This example is basically the same as Example 1, the only difference is that the first set of amplification primers is a sequence of less than or equal to 8 nucleotides added to the 5' end and / or 3' end of B-F1 and B-R1. In this embodiment, one nucleotide is added at the 5' end and 3' end of B-F1 and B-R1 respectively, and the nucleotide can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine), in this embodiment, C is added at the 5' end, G is added at the 3' end, and exons 1-3 of the HLA-B gene can be amplified by using the first set of amplification primers , the second set of amplification primers is the 5' end and / or 3' end of B-F2 and B-R2 to increase the sequence of less than or equal to 8 nucleotides, in this embodiment, respectively in B-F2 and B-R2 - The 5' and 3' ends of R2 increase the sequence of 8 nucleotides. In this example, the 5'→3' at the 5' end increases CACAGTGT, and the 5'→3' at t...
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