Fluorescence quantitative PCR detection kit for beta-thalassemia mutation
A thalassemia detection kit technology, applied in the field of molecular biology, can solve problems such as false positives, difficulty in popularization, poor PCR amplification, etc.
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Embodiment 1
[0083] Example 1: Detection of base deletion mutations (CD41-42 deletion mutations) corresponding to amino acids 41 / 42 of the β-globin gene
[0084] Sample description: Positive samples were collected from the wax block tissue of the specific site mutation of β-thalassemia diagnosed clinically and pathologically in the Department of Pathology, Cancer Hospital Affiliated to Sun Yat-sen University. Genomic DNA was extracted from the wax blocks for the following experimental applications.
[0085] Design a pair of probes and common primers that can specifically detect the base deletion corresponding to amino acid 41 / 42 of the β-globin gene:
[0086] Wherein, the sequence of the PCR forward primer is shown in SEQ ID NO:1, and the sequence of the PCR reverse primer is shown in SEQ ID NO:2:
[0087] A1: 5'-CATAACAGCATCAGGAGTGGACAG-3' (SEQ ID NO: 1);
[0088] A2: 5'-ACTGACTCTCTCTGCCTATTGGTCTATT-3' (SEQ ID NO: 2);
[0089] Fluorescent probes are:
[0090] A3: 5'-HEX-AGGACTCAAAGAAC...
Embodiment 2
[0097] Example 2: Detection of the 654th base mutation (IVS-II-654(C→T)) of the second intron of the β-globin gene
[0098] Design a probe and a pair of common primers that can specifically detect the 654th base mutation of the second intron of the β-globin gene:
[0099]Wherein, the sequence of the PCR forward primer is shown in SEQ ID NO:5, and the sequence of the PCR reverse primer is shown in SEQ ID NO:6:
[0100] B1: 5'-TGGTAGCTGGATTGTAGCTGCTATTA-3' (SEQ ID NO: 5);
[0101] B2: 5'-TTGCACCATTCTAAAGAATAACAGTGA-3' (SEQ ID NO: 6);
[0102] Fluorescent probes are:
[0103] B3: 5'-HEX-TGCTATTGCCTTAACC-MGB-3' (SEQ ID NO: 7);
[0104] B4: 5'-FAM-TTGCTATTACCTTAACCC-MGB-3' (SEQ ID NO: 8);
[0105] Among them, the probe B3 that detects the base corresponding to the 654th position of the second intron of the wild-type β-globin gene: 5'-HEX-TGCTATTGCCTTAACC-MGB-3' (SEQ ID NO: 7), detects β- Probe B4 for the mutation of base 654 in the second intron of the globin gene: 5'-FAM-TTGC...
Embodiment 3
[0110] Example 3: Detection of the base mutation corresponding to the 17th amino acid of the β-globin gene (CD17(A→T))
[0111] Design a pair of probes and common primers that can specifically detect the base mutation corresponding to the 17th amino acid of the β-globin gene:
[0112] The sequence of the PCR forward primer is shown in SEQ ID NO: 9, and the sequence of the PCR reverse primer is shown in SEQ ID NO: 10:
[0113] C1: 5'-CTCCTTAAACCTGTCTTGTAACCTTGATA-3' (SEQ ID NO: 9);
[0114] C2: 5'-TGAGGAGAAGTCTGCCGTTACTG-3' (SEQ ID NO: 10);
[0115] Fluorescent probes are:
[0116] C3: 5'-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3' (SEQ ID NO: 11);
[0117] C4: 5'-FAM-CCACGTTCACCTAGCCCCACAGG-TAMRA-3' (SEQ ID NO: 12);
[0118] Among them, the probe C3 that detects the base corresponding to the 17th amino acid of the wild-type β-globin gene: 5'-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3' (SEQ ID NO: 11), detects the 17th position of the β-globin gene Probe C4 for the base mutation correspon...
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