Xây dựng quy trình phân type rs1801133 dựa trên kỹ thuật real-time HRM=Building a molecular typing protocol for rs1801133 based on real-time PCR HRM technique

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Tác giả: Đăng Khoa Lê, Ngụy Hoàng Linh Lê, Quang Trí Lê, Hoàng Chương Nguyễn, Hoàng Nhất Minh Nguyễn, Thị Huỳnh My Nguyễn, Thị Nhựt Linh Trần

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Phát triển Khoa học & Công nghệ: Chuyên san Khoa học Tự nhiên, 2018

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ID: 472966

Thêm vào giỏ

rs1801133 is a single nucleotide polymorphism (SNP) located in the sequence of MTHFR on human chromosome 1. The alleles of this SNP affect the activity of the MTHFR enzyme. People bearing C/T genotype have 66% activity of MTHFR while people with T/T genotype have only 25% activity. These reduced activities of MTHFR cause homocysteinemia. There are several publications on the relationship between homocysteinemia and human diseases such as cardiovascular disease, neurological diseases, abnormal fetus, infertility and cancer. In this study, we built a molecular protocol for genotyping rs1801133 using real-time PCR HRM technique. This protocol could be used for diagnosis of molecular mechanism of homocysteinemia causing the mentoned above diseases as well as for the study of the relationship between rs1801133 and other human diseases. We successfully designed the primer pairs for genotyping and nucleotide sequencing rs1801133 by real-time PCR HRM and Sanger sequencing method. We also examined the optimal MgCl2 concentration for clear differentiation of three rs1801133 genotypes. Performance characteristics of the real-time PCR HRM protocol included of specificity, repeatability, reproducibility was evaluated and it showed good results. Comparison of genotyping results of rs1801133 between the real-time PCR HRM method and the Sanger nucleotide sequencing method showed good concordances. Finally, this real-time PCR HRM protocol for rs1801133 genotyping was applied on 100 human DNA samples to evaluate the clinical utility of the protocol.rs1801133 là một đa hình đơn nucleotide nằm trên gene MTHFR thuộc nhiễm sắc thể số 1 ở người. Các allene của SNP này hưởng tới hoạt tính của enzyme MTHFR. Người mang genotype C/T của SNP này có hoạt tính enzyme bị giảm còn 66% trong khi người mang genotype T/T có hoạt tính bị giảm còn 25%. Khi hoạt tính enzyme MTHFR giảm sẽ dẫn đến tình trạng gia tăng homocysteine trong máu. Có rất nhiều bằng chứng về sự liên quan giữa tình trạng gia tăng homocysteine và các bệnh ở người như bệnh tim mạch, thần kinh, bất thường thai nhi, vô sinh, ung thư. Trong nghiên cứu này, chúng tôi xây dựng một quy trình phân tử nhằm phân type rs1801133 sử dụng kỹ thuật real-time HRM. Quy trình này có thể được sử dụng để chẩn đoán nguyên nhân phân tử của chứng tăng homocysteine trong máu của các chứng bệnh đã đề cập ở trên. Ngoài ra, nó cũng được sử dụng như một công cụ để nghiên cứu sự liên quangiữa SNP này với các chứng bệnh khác ở người. Chúng tôi đã thiết kế thành công các cặp primer nhằm phân type rs1801133 cũng như để giải trình tự nucleotide SNP này, cũng như đã khảo sát nồng độ MgCl2 thích hợp để phân biệt 3 dạng genotype tương ứng của rs1801133. Khảo sát các đặc tính kỹ thuật của quy trình như độ đặc hiệu kỹ thuật, độ lặp lại, độ tái lập cho kết quả tốt. Cuối cùng, quy trình real-time PCR HRM phân type rs1801133 trên 100 DNA mẫu lâm sàng có so sánh một phần với kỹ thuật giải trình tự nucleotide Sanger. Kết quả thu nhận được cho thấy quy trình phân type hiệu quả rs1801133 được áp dụng trên các mẫu thực tế, vì vậy quy trình này có thể được ứng dụng để phân type rs1801133 trong thực tế lâm sàng.

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