Method used for detecting HLA-B*5801 alleles
A technology of HLA-B and alleles, applied in the field of alleles, can solve the problems of difficult interpretation of primer results, inconvenient result interpretation, increased workload, etc., and achieve accurate and reliable detection results, easy operation, and less interference factors.
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Embodiment 1
[0069] Example 1 Detection of HLA-B*5801 alleles by PCR-SSP method
[0070] 1. Extract DNA:
[0071] After collecting venous blood (2ml) in vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA) according to conventional methods, DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany);
[0072] 2. Design sequence-specific primers and internal reference primers:
[0073]Design three pairs of specific primers for HLA-B*5801 in the concentrated area of HLA-B gene polymorphism sites, the first upstream primer F1: 5'-CCGGGTCCGAGATCCGTCT-3' (sequence 1), the second upstream primer F2 : 5'-GGGCCGGAGTATTGGGATG-3' (SEQ ID NO: 2), the third upstream primer F3: 5'-accgagagaacctgcggat-3' (SEQ ID NO: 3), the universal downstream primer R: 5'-gccatacatcctctggatga-3' (SEQ ID NO: 4); , using the Primer 3 software (http: / / frodo.wi.mit.edu / ) to design internal reference primers on the β-globin gene, the upstream primer Globin-F: 5'- AGTCAGGGCAG...
Embodiment 2
[0079] Example 2 Detection of HLA-B*5801 alleles by Taqman probe method
[0080] 1. Extract DNA:
[0081] After collecting venous blood (2ml) in vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA) according to conventional methods, DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany);
[0082] 2. Design primers and probes:
[0083] Design specific primers for HLA-B*5801 in the polymorphic site concentrated region, upstream primer F2: 5'-GGGCCGGAGTATTGGGATG-3' (SEQ ID NO: 2), downstream primer R: 5'-gccatacatcctctggatga-3' (SEQ ID NO: 4) , and two supporting fluorescent probes: probe1: 5'-CY5-TCCGAGATCCGCCTCCCTGAGGCC-BHQ2-3' (SEQ ID NO: 7) and probe2: 5'-JOE- ACCGAGAGAACCTGCGGATCGCGCTCC-BHQ2-3' (SEQ ID NO: 8); Internal reference primers were designed on the globin gene, upstream primer Globin-F: 5'- AGTCAGGGCAGAGCCATCTA-3' (SEQ ID NO: 5), downstream primer Globin-R: 5'-TTAGGGTTGCCCATAACAGC-3' (SEQ ID NO: 6), and supporting ...
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